16.1 Probes for Following Receptor Binding,
Endocytosis and Exocytosis
The plasma membrane defines the inside and outside of the cell. It not only encloses
the cytosol to maintain the intracellular environment but also serves as a formidable
barrier to the extracellular environment. Because cells require input from their surroundings — in the form of hydrated ions, small polar molecules, large biomolecules and even
other cells — they have developed strategies for overcoming this barrier. Many of these
mechanisms involve initial formation of receptor–ligand complexes, often followed by
transport of the ligand across the cell’s membrane. A useful compendium of human
diseases that affect intracellular transport processes has been published.1
This section focuses on probes for following receptor binding, endocytosis and exocytosis.2 Section 16.2 contains probes for neurotransmitter receptors, which are transmembrane proteins that allow the cell to respond to its environment by selectively binding
ligands and then transmitting this signal across the plasma membrane (Figure 18.1).
Section 16.3 discusses probes for ion channels and carriers, which mediate some signal
transduction processes but also allow the cell to maintain or modify its electrolyte balance and control its osmotic properties.
Ligands for Studying Receptor-Mediated Endocytosis
Molecular Probes offers a variety of fluorescent and fluorogenic ligands that bind to
membrane receptors and, in many cases, are internalized upon binding to their specific
cell receptor. In some cases, the bound ligand is released intracellularly and the receptor
is then recycled to the plasma membrane. Receptor binding may also result in signal
transduction (Chapter 18), Ca2+ mobilization (Chapter 20), intracellular pH changes
(Chapter 21) and formation of reactive oxygen species (ROS, Chapter 19).
Fc OxyBURST Green Assay Reagent: Fluorogenic Immune Complex
Our OxyBURST technology utilizes conjugates of dihydrofluorescein dyes as fluorogenic substrates that can easily detect receptor-mediated uptake of biomolecules during
phagocytosis. When soluble or surface-bound IgG immune complexes interact with Fc
receptors on phagocytic cells, a number of host defense mechanisms are activated, including phagocytosis and activation of an NADPH oxidase–mediated oxidative burst.3 Dichlorodihydrofluorescein diacetate (H2DCFDA, D-399; Section 19.2; Figure 19.6), a cellpermeant fluorogenic probe that localizes in the cytosol, has frequently been used to
monitor this oxidative burst; 4 however, its fluorescence response is limited by the diffusion rate of the reactive oxygen species into the cytosol from the phagovacuole where it is
generated. In contrast, our Fc OxyBURST assay reagents permit direct measurement of
the kinetics of Fc receptor–mediated internalization and the subsequent oxidative burst in
the phagovacuole, yielding signals that are many times brighter than those generated
by H2DCFDA.
Molecular Probes’ Fc OxyBURST Green assay reagent (F-2902) was developed in
collaboration with Elizabeth Simons of Boston University to monitor the oxidative burst
in phagocytic cells using fluorescence instrumentation. The Fc OxyBURST Green assay
reagent comprises bovine serum albumin (BSA) that has been covalently linked to
dichlorodihydrofluorescein (H2DCF) and then complexed with a purified rabbit polyclonal anti-BSA antibody (our A-11133). When these immune complexes bind to Fc
receptors, the nonfluorescent H2DCF molecules are internalized within the phagovacuole
and subsequently oxidized to green-fluorescent 2′,7′-dichlorofluorescein (DCF, Figure
16.1; Figure 16.2). Unlike H2DCFDA, the Fc OxyBURST Green assay reagent does not
require intracellular esterases for activation, making this reagent particularly suitable for
detecting the oxidative burst in cells with low esterase activity such as monocytes.5 The
Fc OxyBURST Green assay reagent reportedly produces >8 times more fluorescence
than does H2DCFDA at 60 seconds and >20 times more at 15 minutes following internalization of the immune complex.6
Figure 16.1 Fluorescence emission of human neutrophils challenged either with Molecular Probes’
Fc OxyBURST Green assay reagent (H2DCF-BSA
immune complexes, F-2902) or with unlabeled immune complexes in the presence of dichlorodihydrofluorescein diacetate (H2DCFDA, D-399; Section
19.2). The Fc OxyBURST Green assay reagent generates significantly more fluorescence than does
the more commonly used H2DCFDA. Flow cytometry data provided by Elizabeth Simons, Boston University (J Immunol Methods 130, 223 (1990)).
Figure 16.2 Fc OxyBURST reagent (F-2902) for
fluorescent detection of the Fc receptor–mediated
phagocytosis pathway. Dichlorodihydrofluorescein
(H2DCF) is covalently attached to bovine serum albumin (BSA), then complexed with a rabbit polyclonal anti-BSA antibody (A-11133). Upon binding
to an Fc receptor, the nonfluorescent immune
complex is internalized and subsequently oxidized
to the fluorescent DCF.
Section 16.1
673
Various reports have described the use of the Fc OxyBURST Green assay reagent to
study the oxidative burst in phagovacuoles.7,8 Neutrophils from patients with chronic
granulomatous disease, a genetic deficiency known to disable NADPH oxidase–mediated
oxidative bursts, were observed to bind but not oxidize the Fc OxyBURST Green assay
reagent 6 (Figure 16.3). Using microfluorometry to detect the Fc OxyBURST Green
response, researchers were able to simultaneously monitor oxidative activity and membrane currents in voltage-clamped human mononuclear cells.9 The Fc OxyBURST Green
assay reagent has also been employed to assess the effect of manganese-based superoxide
dismutase mimetics on superoxide generation in human neutrophils.10
Figure 16.3 Oxidative bursts of human neutrophils
from a healthy donor (control) compared to those
from a patient with chronic granulomatous disease
(CGD), as detected using the Fc OxyBURST Green
assay reagent (F-2902). Flow cytometry data
provided by Elizabeth Simons, Boston University
(J Immunol Methods 130, 223 (1990)).
Figure 16.4 OxyBURST Green H2HFF BSA reagent
(O-13291) detecting leakage of hydrogen peroxide
(H2O2) from cells. In the presence of horseradish
peroxidase (HRP) and H2O2, the nonfluorescent
OxyBURST Green H2HFF BSA reagent is oxidized to
a green-fluorescent product.
Distributors
Molecular Probes sells its products
directly in most countries. Current
exceptions include Japan, Korea,
Australia and Mexico, where Molecular
Probes requests that customers order
from one of our nonexclusive distributors. A list of all of our current nonexclusive distributors starts on page iv
of this Handbook. This list is subject to
change. The terms and prices offered
by our distributors may differ from the
terms offered by Molecular Probes.
Full contact information for our distributors and links to their Web sites
appears at our Web site
(www.probes.com/distributors).
674
OxyBURST Green H2HFF BSA Reagent
The OxyBURST Green H2HFF BSA reagent (O-13291) is similar to the Fc OxyBURST
reagent, except that it is prepared by reacting the succinimidyl ester of a reduced form of
our Oregon Green 488 dye with BSA. The absorption maximum (~492 nm) of the oxidation product of this reagent is better matched to the 488 nm line of the argon-ion laser than
is that of the Fc OxyBURST Green assay reagent. We have utilized the OxyBURST Green
H2HFF BSA reagent in combination with horseradish peroxidase to detect leakage of
hydrogen peroxide from cells (Figure 16.4). The OxyBURST Green H2HFF BSA reagent
can also be complexed with anti-BSA antibody (see below) to form an immune complex
that can be utilized like the Fc OxyBURST Green assay reagent (F-2902, see above).
All of the OxyBURST reagents are slowly oxidized by molecular oxygen and are also
susceptible to oxidation catalyzed by illumination in a fluorescence microscope. These
reagents are reasonably stable in solution for at least six months when stored under nitrogen or argon in the dark at 4°C. We also offer a purified rabbit polyclonal anti-BSA antibody (A-11133), which can bind any of our fluorescent BSA conjugates (Section 14.7) or
fluorogenic DQ BSA conjugates (D-12050, D-12051; Section 10.4) to create immune
complexes for analyzing the Fc receptor–mediated phagocytosis pathway. In the case of
the anti-BSA complex with DQ BSA, initial binding and internalization of the probe is
followed by hydrolysis to fluorescent peptides within the phagovacuole (Figure 16.5).
Amine-Reactive OxyBURST Green Reagents
As an alternative to our Fc OxyBURST Green assay reagent and OxyBURST Green
H2HFF BSA, Molecular Probes offers amine-reactive OxyBURST Green H2DCFDA
succinimidyl ester (2′,7′-dichlorodihydrofluorescein diacetate, SE; D-2935; Figure 16.6),
which can be used to prepare oxidation-sensitive conjugates of a wide variety of biomolecules and particles, including antibodies, antigens, peptides, proteins, dextrans, bacteria,
yeast and polystyrene microspheres.6,11 Following conjugation to amines, the two acetates of the OxyBURST Green H2DCFDA reagent can be removed by treatment with
hydroxylamine at near-neutral pH to yield the dichlorodihydrofluorescein conjugates.
The OxyBURST Green H2DCFDA conjugates are nonfluorescent until they are oxidized
to the corresponding fluorescein derivatives. Thus, like our Fc OxyBURST Green assay
reagent, they provide a means of detecting the oxidative burst in phagocytic cells. In one
application, OxyBURST Green H2DCFDA succinimidyl ester was conjugated to an
antibody that binds specifically to YAC tumor cells. YAC cells opsonized with this customized OxyBURST reagent were then used in a fluorescence microscopy study to show
that Fc receptor–activated neutrophils appear to deliver reactive oxygen species to the
surface of their target cells.11
Several other reagents — dihydrofluoresceins, dihydrorhodamines, dihydroethidium
and chemiluminescent probes — that have been used to detect the reactive oxygen species (ROS) produced during phagocytosis are described in Section 19.2.
Fluorescent Low-Density Lipoprotein Complexes
The human LDL complex, which delivers cholesterol to cells by receptor-mediated
endocytosis, comprises a core of about 1500 molecules of cholesteryl ester and triglyceride, surrounded by a 20 Å–thick shell of phospholipids, unesterified cholesterol and a
single copy of apoprotein B 100 (MW ~500,000 daltons).12 Once internalized, LDL
dissociates from its receptor and eventually appears in lysosomes.13 Molecular Probes
offers unlabeled LDL (L-3486), which has been reported to be an effective vehicle for
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
selectively delivering antitumor drugs to cancer cells.14 We also prepare two classes of
labeled LDL probes — those containing an unmodified apoprotein, used to study the
mechanisms of normal cholesterol delivery and internalization, and those with an acetylated apoprotein, used to study endothelial, microglial and other cell types that express
receptors that specifically bind this modified LDL (see below).
Molecular Probes offers LDL labeled with either DiI (DiI LDL, L-3482) or the
BODIPY FL fluorophore (BODIPY FL LDL, L-3483), highly fluorescent lipophilic
dyes that diffuse into the hydrophobic portion of the LDL complex without affecting the
LDL-specific binding of the apoprotein. The contrasting fluorescence of DiI LDL and
fluorescein transferrin (T-2871) permits their simultaneous use to follow the lysosomally
directed pathway and the recycling pathways, respectively.15 As compared to DiI LDL,
BODIPY FL LDL is more efficiently excited by the 488 nm spectral line of the argon-ion
laser, making it better suited for flow cytometry and confocal laser-scanning microscopy
studies. Like our BODIPY FL C5-ceramide and its BSA complex (D-3521, B-22650,
Section 12.4), BODIPY FL LDL fluoresces somewhat in the red region, particularly at
high concentration or as aggregates (Figure 13.6), sometimes precluding its use for multicolor labeling with red fluorophores. Both BODIPY FL LDL and DiI LDL have been
used to investigate the binding specificity and partitioning of LDL throughout the Schistosoma mansoni parasite 16 (Figure 16.7). Fluorescent LDL complexes have also proven
useful in a variety of experimental systems to:
• Count the number of cell-surface LDL receptors, analyze their motion and clustering
and follow their internalization 17–19
• Demonstrate that fibroblasts grown continuously in the presence of DiI LDL (L-3482)
proliferate normally and exhibit normal morphology,20 making DiI LDL a valuable
alternative to 125 I-labeled LDL for quantitating LDL receptor activity 21
• Identify LDL receptor–deficient Chinese hamster ovary (CHO) cell mutants 22
• Investigate the expression of LDL receptors in granulosa and luteal cells in primate
and porcine ovarian follicles 23–25
• Track the mobility of LDL receptors in an electric field 26–28
Figure 16.5 Immune complex of DQ BSA conjugate (D-12050, D-12051) with an anti–bovine
serum albumin (BSA) antibody (A-11133) for the
fluorescent detection of the Fc receptor–mediated
phagocytosis pathway. The DQ BSA is a derivative
of BSA that is labeled to such a high degree with
either the green-fluorescent BODIPY FL or redfluorescent BODIPY TR-X dyes, that the dyes are
self-quenched. Upon binding to an Fc receptor,
the nonfluorescent immune complex is internalized and the protein is subsequently hydrolyzed to
fluorescent peptides within the phagovacuole.
We prepare our fluorescent LDL products from fresh human plasma for sale at the end
of every odd month, and ship them within two weeks of their preparation. If stored refrigerated and protected from light, our LDL products are stable for at least four to six weeks
from the date of shipment; these products must not be frozen. Because preparation of
these complexes involves several variables, some batch-to-batch variability in degree of
labeling and fluorescence yield is expected.
Fluorescent Acetylated LDL Complexes
If the lysine residues of LDL’s apoprotein have been acetylated, the LDL complex no
longer binds to the LDL receptor,29 but rather is taken up by macrophage and endothelial
cells that possess “scavenger” receptors specific for the modified LDL.30,31 Once the
acetylated LDL (AcLDL) complexes accumulate within these cells, they assume an
appearance similar to that of foam cells found in atherosclerotic plaques.32–34 Molecular
Probes offers AcLDL labeled with DiI (DiI AcLDL, L-3484; Figure 16.8), the Alexa
Fluor 488 fluorophore (Alexa Fluor 488 AcLDL, L-23380) or the BODIPY FL fluorophore (BODIPY FL AcLDL, L-3485). Fluorescent dye conjugates of high-density lipoproteins, including one prepared using our Alexa Fluor 488 succinimidyl ester (A-20000,
A-20100; Section 1.3), are taken up via the same receptor.35
Using DiI AcLDL, researchers have discovered that the scavenger receptors on rabbit
fibroblasts and smooth muscle cells appear to be up-regulated through activation of the
protein kinase C pathway.36 DiI AcLDL has also been used to show that Chinese hamster
ovary (CHO) cells express AcLDL receptors that are distinct from macrophage scavenger
receptors.37,38 Ultrastructural localization of endocytic compartments that maintain a
connection to the extracellular space has been achieved by photoconversion of DiI
AcLDL in the presence of diaminobenzidine 39 (see Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents in Section 1.5). A quantitative assay for LDL- and
scavenger-receptor activity in adherent and nonadherent cultured cells that avoids the use
of both radioactivity and organic solvents has been described.40
Figure 16.6 D-2935 2′,7′-dichlorodihydrofluorescein diacetate, succinimidyl ester (OxyBURST
Green H2DCFDA, SE).
Extra Copies of this
Handbook
Publication of this Handbook involves
considerable effort and expense on the
part of Molecular Probes. Single copies
are provided free upon request to new
customers. Additional copies can be
ordered from Molecular Probes under
catalog number H-19999, or requested
from our distributors (see page iv).
Customers in Japan, Korea, Australia
and Mexico must request copies of the
Handbook from one of our distributors
in those countries.
Section 16.1
675
Figure 16.7 DiI LDL (L-3482) bound to the surface
and internalized in the gut of the parasite Schistosoma mansoni. The distribution of LDL in the parasite is used to study a putative mechanism by
which the parasite may avoid host immune recognition. Image contributed by John P. Caulfield, Harvard School of Public Health.
Figure 16.8 Microglial cells in a rat hippocampus
cryosection labeled with red-orange–fluorescent
DiI acetylated low-density lipoprotein (L-3484)
and stained using blue-fluorescent DAPI (D-1306,
D-3571, D-21490).
It has now become routine to identify endothelial cells and microglial cells in primary
cell culture by their ability to take up DiI-labeled acetylated LDL; 41–44 DiI AcLDL is
taken up by microglia but not by astrocytes.45 DiI AcLDL was employed in order to
confirm endothelial cell identity in investigations of shear stress 46 and P-glycoprotein
expression,47 as well as to identify blood vessels in a growing murine melanoma.48 In
addition, patch-clamp techniques have been used to investigate membrane currents in
mouse microglia, which were identified both in culture and in brain slices by their staining with DiI AcLDL.49,50 For some applications, our Alexa Fluor 488 or BODIPY FL
AcLDL may be the preferred probes because the dyes are covalently bound to the modified apoprotein portion of the LDL complex and are therefore not extracted during
subsequent manipulations of the cells. Furthermore, the Alexa Fluor 488 AcLDL conjugate has spectral characteristics similar to fluorescein (Figure 7.54); however, the Alexa
Fluor 488 dye produces brighter and more photostable conjugates that researchers may
find useful for analyses with instruments equipped with the 488 nm argon-ion laser
excitation sources, including flow cytometers and confocal laser-scanning microscopes.
Fluorescent Conjugates of Lipopolysaccharides
Molecular Probes offers several fluorescent conjugates of lipopolysaccharides (LPS)
from Escherichia coli and Salmonella minnesota (Table 16.1). LPS or endotoxins are
complex macromolecules present on the outer cell walls of gram-negative bacteria. Recognition of LPS by binding to the CD14 cell-surface receptor of phagocytes is the key
initiation step in the mammalian immune response to infection by gram-negative bacteria. The structural core of LPS, and the primary determinant of its biological activity, is
the N-acetylglucosamine derivative, lipid A (Figure 16.9). Two plasma proteins — LPSbinding protein (LBP) and soluble CD14 (sCD14) — play primary roles in transporting
LPS and mediating cellular responses.51–56 If the fatty acid residues are removed from
the lipid A component, the toxicity of the LPS can be reduced significantly. However,
the mono- or diphosphoryl forms of lipid A are inherently toxic. In many gram-negative
bacterial infections, LPS are responsible for clinically significant symptoms like fever,
low blood pressure and tissue edema, which can lead to disseminated intravascular coagulation, organ failure and death. Studies also clearly indicate that LPS induce various
signal transduction pathways, including those involving protein kinase C 57,58 and protein
myristylation,59 and stimulate a variety of immunochemical responses, including B lymphocyte 60 and G-protein activation.61
Our fluorescent LPS allow researchers to follow binding, transport and cell internalization processes of LPS 62 (Figure 16.10). Lipopolysaccharide internalization activates
endotoxin-dependent signal transduction in cardiomyocytes.63 Aggregation of the
BODIPY FL derivative of S. minnesota strain R595 in aqueous solution is reported to
result in a spectral shift of the BODIPY FL dye’s emission maximum to ~620 nm. Addition of LBP results in a relatively small fluorescence increase, indicating that the LPS
remain aggregated upon binding. Subsequent addition of sCD14 results in a large fluorescence intensity increase and an emission peak shift to ~510 nm, indicating LBP-mediated
transfer of LPS monomers to sCD14.53 In one study, a BODIPY FL derivative of LPS
from E. coli strain LCD25 was used to measure the transfer rate of LPS from monocytes
to high-density lipoprotein 64 (HDL). Another study utilized a BODIPY FL derivative of
LPS from S. minnesota to demonstrate transport to the Golgi apparatus in neutrophils,51,52
although this could have been due to probe metabolism. It has been reported that or-
Table 16.1 Fluorescent lipopolysaccharide conjugates.
Figure 16.9 Structure of the lipid A component of
Salmonella minnesota lipopolysaccharide.
Fluorophore
Alexa Fluor 488
Abs *
Em *
Escherichia coli
Salmonella minnesota
495
519
L-23351
L-23356
BODIPY FL
503
513 †
L-23350
L-23355
Alexa Fluor 568
578
603
L-23352
L-23357
Alexa Fluor 594
590
617
L-23353
L-23358
* Approximate absorption (Abs) and fluorescence emission (Em) maxima for conjugates, in nm. † At high
concentrations, such as in micelles or aggregates, the emission maximum for BODIPY FL will shift from
~513 nm to ~620 nm (J Immunol 158, 3925 (1997), J Biol Chem 271, 4100 (1996)).
676
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
ganelles other than the Golgi are labeled by some fluorescent or
nonfluorescent LPS.65,66 Cell-surface–bound fluorescent LPS can
be quenched by trypan blue 64 (Figure 16.23). Molecular Probes’
fluorescent LPS can potentially be combined with other fluorescent indicators, such as Ca2+-, pH- or organelle-specific stains, to
monitor intracellular localization, as well as real-time changes in
cellular response to LPS. Cationic lipids are reported to assist the
translocation of fluorescent lipopolysaccharides into live cells.67
We also offer the Pro-Q Emerald 300 Lipopolysaccharide Gel
Stain Kit (P-20495, Section 3.2, Figure 3.19, Figure 3.20), which
permits ultrasensitive analysis of unlabeled lipopolysaccharides
in gel electrophoretograms (Figure 3.18).
Epidermal Growth Factor Derivatives
Epidermal growth factor (EGF) is a 53–amino acid polypeptide hormone (MW 6045 daltons) that stimulates division of
epidermal and other cells.68–70 The EGF receptors include the
HER-2/neu receptor (where “HER-2” is an acronym for human
epidermal growth factor receptor-2 and “neu” refers to an original
mouse origin); HER-2/neu overexpression has evolved as a prognostic/predictive factor in some solid tumors. Binding of EGF to
its 170,000-dalton receptor protein results in the activation of
kinases, phospholipases and Ca2+ mobilization and precipitates a
wide variety of cellular responses related to differentiation, mitogenesis, organ development and cell motility.
Molecular Probes offers unlabeled mouse submaxillary gland
EGF (E-3476), as well as EGF labeled with biotin-XX, fluorescein, Oregon Green 514 and tetramethylrhodamine (E-3477,
E-3478, E-7498, E-3481), all containing a single biotin or fluorophore on the N-terminal amino acid. The long spacer arm in our
biotin-XX EGF enhances the probe’s affinity for the EGF receptor.71 The dissociation constant of the EGF conjugates in DMEMHEPES medium is in the low nanomolar range for human epidermoid carcinoma (A431) cells,72 a value that approximates that of
the unlabeled EGF.
Figure 16.10 Flow cytometric analysis of blood using Alexa Fluor 488 lipopolysaccharide (LPS).
Human blood was incubated with Alexa Fluor 488
LPS from Escherichia coli (L-23351) and antiCD14 antibody on ice for 20 minutes. The red
blood cells were lysed and the sample was analyzed on a flow cytometer equipped with a 488 nm
Ar–Kr excitation source and a 525 ± 12 nm bandpass emission filter. Monocytes were identified on
the basis of light scatter, CD14 expression and
Alexa Fluor 488 LPS binding.
Fluorescently labeled EGF has enabled scientists to use fluorescence resonance energy transfer techniques to assess EGF
receptor–receptor and receptor–membrane interactions 73–75 (see
Fluorescence Resonance Energy Transfer (FRET) in Section 1.3).
Using fluorescein EGF as the donor and tetramethylrhodamine
EGF as the acceptor, researchers examined temperature-dependent lateral and transverse distribution of EGF receptors in A431
cell plasma membranes.75 When fluorescein EGF binds to A431
cells, it apparently undergoes a biphasic quenching, which can
be attributed first to changes in rotational mobility upon binding
and then to receptor–ligand internalization. By monitoring this
quenching in real time, the rate constants for the interaction of
fluorescein EGF with its receptor were determined.76
Although fluorescently labeled EGF can be used to follow
lateral mobility and endocytosis of the EGF receptor,77,78 the
visualization of fluorescent EGF may require low-light imaging
technology, especially in cells that express low levels of the EGF
receptor.79 In cells with few EGF receptors, it can be difficult to
detect signal over background fluorescence unless signal amplification methods are employed. Oregon Green 514 dye–labeled
EGF is one of our most fluorescent and photostable EGF conjugates. We also prepare biotinylated EGF complexed to Alexa
Fluor 488 streptavidin (E-13345, Figure 16.11) and to Texas Red
streptavidin 80,81 (E-3480). These products yield severalfold
brighter signals per EGF receptor when compared with the direct
conjugates. We have found that EGF receptors can easily be
detected with these complexes without resorting to low-light
imaging technology (Figure 16.12). We have also used our avidin- and streptavidin-coated FluoSpheres and TransFluoSpheres
microspheres (Section 6.5) for the sensitive detection of EGF
receptors by flow cytometry.82
In addition, we offer biotin-XX EGF, which contains a long
spacer arm that facilitates binding of fluorescent or enzymeconjugated streptavidins (Section 7.6). Using biotinylated EGF
and phycoerythrin-labeled secondary reagents (Section 6.4),
Figure 16.11 Early endosomes in live HeLa cells
identified after a 10-minute incubation with greenfluorescent Alexa Fluor 488 epidermal growth factor (E-13345). The cells were subsequently fixed
with paraformaldehyde and labeled with an antibody to the late endosomal protein, RhoB. That
antibody was visualized with a red-orange–fluorescent secondary antibody. Nuclei were stained
with TO-PRO-3 iodide (T-3605, pseudocolored
blue). Image contributed by Harry Mellor, University of Bristol.
Figure 16.12 Lightly fixed human epidermoid carcinoma cells (A431) stained with Molecular
Probes’ biotinylated epidermal growth factor
(EGF) complexed to Texas Red streptavidin
(E-3480). An identical cell preparation stained in
the presence of 100-fold excess unlabeled EGF
(E-3476) showed no visible fluorescence.
Section 16.1
677
researchers were able to detect as few as 10,000 EGF cell-surface receptors by confocal
laser-scanning microscopy.83 We have used our ELF 97 Cytological Labeling Kit
(E-6603, Section 6.3) in conjunction with biotinylated EGF to detect low-abundance
EGF receptors (Figure 6.24). Tyramide signal-amplification (TSA) technology (Section
6.2) is particularly valuable for detection and localization of low-abundance EGF receptors by both imaging and flow cytometry (Figure 6.11).
Transferrin Conjugates
Transferrin is a monomeric serum glycoprotein (MW ~80,000 daltons) that binds up
to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis.84–86 Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of the sequestered iron from the transferrin–receptor complex.
Following the release of iron, the apotransferrin is recycled to the plasma membrane,
where it is released from its receptor to scavenge more iron.87,88 Labeled transferrin
conjugates can therefore be used with fluorescent LDL to distinguish the lysosomally
directed and recycling endosomal pathways.13,15
Molecular Probes’ fluorescent di-ferric (Fe3+) human transferrin conjugates include
those of the fluorescein, BODIPY FL,89 Oregon Green 488,90 Alexa Fluor 488,91–94 Alexa
Fluor 546, Alexa Fluor 568, Alexa Fluor 594 95–97 (Figure 16.13), Alexa Fluor 647, tetramethylrhodamine and Texas Red fluorophores (Table 16.2). Fluorescently labeled transferrin has greatly aided the investigation of endocytosis.98–100 The Alexa Fluor conjugates
of transferrin 92–97,101,102 are highly recommended because of their brightness, enhanced
photostability and lack of sensitivity to pH (Section 1.3). The pH sensitivity of fluorescein-labeled transferrin has been exploited to investigate events occurring during endosomal acidification.103–106 Fluorescent transferrins have also been used to:
Figure 16.13 Live HeLa cells incubated with Alexa
Fluor 594 transferrin (T-13343) for 10 minutes to
label early endosomes. The cells were subsequently fixed with paraformaldehyde and labeled with an
antibody to the endosomal protein RhoD. That
antibody was visualized with a green-fluorescent
secondary antibody. Yellow fluorescence indicates
regions of co-localization. Image contributed by
Harry Mellor, University of Bristol.
• Analyze the role of the γ-chain of type III IgG receptors in antigen–antibody complex
internalization 107
• Define the nature of several mutations that affect the endosomal pathway 108–110
• Demonstrate that the fungal metabolite brefeldin A (B-7450, Section 12.4) induces
an increase in tubulation of transferrin receptors in BHK-21 cells 111 and in the
perikaryal–dendritic region of cultured hippocampal neurons 112
• Observe receptor trafficking in living cells by confocal laser-scanning microscopy and
show that recycling transferrin receptors are distributed on the surface of the leading
lamella in migrating fibroblasts 113
• Show that the endosomal compartment of living epidermoid carcinoma cells is an
extensive network of tubular cisternae 114
Table 16.2 Transferrin conjugates.
Cat #
Figure 16.14 Crystal structure of recombinant human
Endostatin protein (E-23377). Image courtesy of
Entremed, Inc.
Label
Abs *
Em *
T-23363
biotin-XX
NA
NA
T-2871
fluorescein
494
518
T-13342
Alexa Fluor 488
495
518
T-13341
Oregon Green 488
496
524
T-2873
BODIPY FL
505
513
T-2872
tetramethylrhodamine
555
580
T-23364
Alexa Fluor 546
556
575
T-23365
Alexa Fluor 568
578
603
T-13343
Alexa Fluor 594
589
616
T-2875
Texas Red
595
615
T-23362
Alexa Fluor 633
632
647
T-23366
Alexa Fluor 647
650
665
* Approximate absorption (Abs) and fluorescence emission (Em) maxima for conjugates, in nm. NA = Not
applicable.
678
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
Uptake of a horseradish peroxidase (HRP) conjugate of transferrin by endosomes has been detected using tyramide signal amplification (TSA, Section 6.2) by catalytic deposition of biotin tyramide
and use of fluorescent streptavidin conjugates 115 (Section 7.6).
In addition to its fluorescent and biotinylated transferrin conjugates, Molecular Probes carries a mouse IgG1 monoclonal anti–
human transferrin receptor antibody (A-11130). It should be
possible to combine this antibody with any of our Zenon One
reagents (Section 7.2, Table 7.1) for rapid preparation of labeling
complexes (Figure 7.32). Antibodies against transferrin receptors
have been used for indirect immunofluorescence staining of the
transferrin receptor,116–118 transport of molecules across the
blood–brain barrier,119 the characterization of transferrin in recycling compartments,116 enzyme-linked immunosorbent assays
(ELISAs) 118 and antibody competition with transferrin uptake.120
Fluorescent Lactoferrin
Lactoferrin, a member of the transferrin family of bilobal,
monomeric glycoproteins 85 (MW ~80,000 daltons), possesses
antiviral and antimicrobial activity and is found in milk, exocrine
fluids and leukocytes. Lactoferrin functions as a regulatory protein, stimulating phagocytic and cytotoxic functions of macrophages, as well as influencing normal and tumor cell growth.121
Lactoferrin receptors exist in a wide variety of cells, including
activated hepatocytes,122 T lymphocytes,123 mammary epithelial
cells,124 bacteria,125–129 rat liver,130 various types of brain cells,131
and sperm,132 in which it forms a major component of spermcoating antigens. Lactoferrin binding and transport in Streptococcus uberis is unaffected by the presence of bovine transferrin.133
The number of lactoferrin receptors is increased in patients with
Parkinson’s disease.134–136 A conjugate produced by linking a
reactive fluorophore to carbohydrate moieties on the lactoferrin
was used to show that receptor-mediated binding of lactoferrin
can inhibit platelet aggregation.137 The parasitic protozoan Tritrichomonas foetus selectively binds and internalizes lactoferrin
conjugates.138,139 Lactoferrin (but not transferrin) binds to DNA
and CpG motifs of oligodeoxynucleotides.140 Our lactoferrin
conjugate (L-13350) is prepared by linking the Oregon Green 514
dye directly to lysine residues of the protein.
Fluorescent Fibrinogen
Fibrinogen is a key component in the blood clotting process
and can support both platelet–platelet and platelet–surface interactions by binding to the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor (also called integrin αIIbβ3) of activated platelets.141–145 Although not well understood, activation of GPIIb-IIIa is required
for fibrinogen binding, which leads to platelet activation, adhesion, spreading and microfilament reorganization of human endothelial cells in vitro.141,144,146,147 Bone marrow transplant patients have significantly higher levels of fibrinogen binding as
compared to controls.148 Soluble fibrinogen binds to its receptor
with a Ca2+-dependent apparent Kd of 0.18 µM.149 This binding is
apparently mediated by the tripeptide sequence Arg–Gly–Asp
(RGD), found in both fibrinogen and fibronectin.144,150,151
Fluorescently labeled fibrinogen has proven to be a valuable
tool for investigating platelet activation and subsequent fibrinogen binding. Fluorescein fibrinogen has been used to identify
activated platelets by flow cytometry.146,152–155 The binding of
fluorescein fibrinogen to activated platelets has been shown to be
saturable and can be inhibited completely by underivatized fibrinogen.154,155 The preferential binding and accumulation of fluorescein fibrinogen at the endothelial border of venular blood vessels
has been studied by quantitative fluorescence microscopy.156 A
biotinylated fibrinogen derivative is rapidly internalized by activated platelets.157
Molecular Probes offers four human fibrinogen conjugates, in
three fluorescent colors. The Alexa Fluor 488 human fibrinogen
conjugate (F-13191) and Oregon Green 488 human fibrinogen
conjugate (F-7496) have spectral characteristics similar to fluorescein conjugates. However, the fluorescence of the Alexa Fluor
488 158 and Oregon Green 488 conjugates is more photostable and
less pH dependent than that of fluorescein conjugates (Figure
1.11, Figure 1.42). The red-orange–fluorescent Alexa Fluor 546
human fibrinogen conjugate (F-13192) is brighter and more
photostable than the spectrally similar tetramethylrhodamine
fibrinogen conjugates. Similarly, the red-fluorescent Alexa Fluor
594 human fibrinogen conjugate (F-13193) is brighter than the
Texas Red fibrinogen conjugate, yet has similar excitation and
emission maxima. These highly fluorescent fibrinogen conjugates
are useful for investigating platelet activation and subsequent
fibrinogen binding using fluorescence microscopy or flow cytometry (Figure 15.82).
Endostatin Protein
The angiogenesis inhibitor, endostatin, is an endogenous
20,000-dalton carboxyl-terminal fragment of collagen XVIII
(Figure 16.14) that induces regression of tumors in mice. Although its complete mechanism of action is unknown, research
has found that endostatin appears to trigger suppression of both
apoptosis factors and cell-proliferation genes, including c-myc
gene expression, resulting in a potent antimigratory effect.159
On the cell surface, endostatin has been shown to bind to glypicans or transmembrane heparan sulfate proteoglycans.160,161 Once
endocytosed, the protein has been demonstrated to bind to tropomyosin,162 a protein that stabilizes the pointed end of actin filaments by slowing depolymerization.163 Indirectly, endostatin
appears to induce increases in cytosolic Ca2+ and cAMP.159 Molecular Probes offers recombinant Endostatin protein (E-23377)
for research purposes; recombinant Angiostatin protein, another
protein useful for angiogenesis research, is also available
(A-23375, Section 15.4).
Fluorescent Gelatin and Type IV Collagen
Collagen is a major component of the extracellular matrix and,
in vertebrates, constitutes approximately 25% of total protein.
This important protein not only serves a structural role, but also is
important in cell adhesion and migration. Specific collagen receptors, fibronectin and a number of other proteins involved in cell–
cell and cell–surface adhesion have been demonstrated to bind
collagen and gelatin (denatured collagen).164,165
Molecular Probes prepares fluorescent conjugates of gelatin
and type IV collagen, the principal collagen in basement membranes. These reagents are designed for researchers studying
collagen-binding proteins and collagen metabolism, as well as
gelatinases and collagenases, which are metalloproteins that
digest gelatin and collagen. We offer two green-fluorescent
Section 16.1
679
gelatin conjugates — fluorescein gelatin and Oregon Green 488
gelatin (G-13187, G-13186). When compared with the fluorescein conjugate, the Oregon Green 488 conjugate exhibits almost
identical fluorescence spectra but its fluorescence is much more
photostable and much less pH dependent (Figure 1.11). For
researchers requiring a more specific substrate, we also offer
the Oregon Green 488 conjugate of human type IV collagen
(C-13185). These highly fluorescent gelatin and collagen conjugates have been shown to be useful for:
• Following integrin-mediated phagocytosis 166
• Localizing surface fibronectin on cultured cells 167
• Studying fibronectin–gelatin interactions in solution using
fluorescence polarization (FP, see Section 1.4) 165,168
Molecular Probes has also developed fluorogenic gelatinase
and collagenase substrates — DQ gelatin and DQ collagen —
which are described in Section 10.4. In addition, we offer fluorescent microspheres coated with collagen, which are described
below. Molecular Probes also has available highly purified rabbit
polyclonal antibodies to matrix metalloproteinases, including
MMP-1 (interstitial collagenase), MMP-2 (gelatinase A or type
IV collagenase), MMP-3 (stromelysin) and MMP-9 (gelatinase B
or type V collagenase). See Section 7.5 and Table 7.16 for more
information on these antibodies.
DQ Ovalbumin: A Probe for Antigen Processing and Presentation
Although antigen processing and presentation have been
extensively studied, the exact sequence and detailed pathways for
generating antigenic peptides have yet to be elucidated. In general, the immunogenic protein is internalized by a macrophage,
denatured, reduced and proteolyzed, and then the resulting peptides associate with MHC class II molecules that are expressed at
the cell surface.169 Ovalbumin is efficiently processed through
mannose receptor–mediated endocytosis by antigen-presenting
cells and is widely used for studying antigen processing.170–172
DQ ovalbumin 173 (D-12053) is Molecular Probes’ self-quenched
ovalbumin conjugate designed specifically for the study of macrophage-mediated antigen processing in flow cytometer– and
microscope-based assays.
Figure 16.15 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and
almost totally quenched substrates provided in our EnzChek Protease Assay
Kits (E-6638, E-6639), EnzChek Amylase Assay Kit (E-11954),
EnzChek Gelatinase/Collagenase Assay Kit (E-12055), EnzChek Elastase Kit
680
Traditionally, fluorescein-labeled bovine serum albumin (FITCBSA) has been used as a fluorogenic protein antigen for studying
the real-time kinetics of antigen processing in live macrophages
by flow cytometry,174 two-photon fluorescence lifetime imaging
microscopy (FLIM) 175 and fluorescence polarization.174,176,177
FITC-ovalbumin has been employed to study antigen uptake in
HIV-1–infected monocytic cells.178 The FITC-ovalbumin and
FITC-BSA used in these experiments were heavily labeled with
fluorescein such that the intact conjugates were relatively nonfluorescent due to self-quenching. Upon denaturation and proteolysis, however, these FITC conjugates became highly fluorescent,
allowing researchers to monitor intracellular trafficking and the
processing of ovalbumin and BSA in macrophages.
For studies of antigen processing and presentation, Molecular
Probes’ DQ ovalbumin offers several advantages when compared
to FITC-ovalbumin and FITC-BSA. Like the FITC conjugates,
DQ ovalbumin is labeled with our pH-insensitive, green-fluorescent BODIPY FL dye such that the fluorescence is almost completely quenched until the probe is digested by proteases (Figure
16.15). But unlike fluorescein, which has greatly reduced fluorescence intensity at acidic pH and is not very photostable, our
BODIPY FL dye exhibits bright, relatively photostable and totally pH-insensitive fluorescence from pH 3 to 9. Furthermore, the
intact DQ ovalbumin is more highly quenched than unprocessed
FITC-ovalbumin or FITC-BSA at a lower degree of substitution,
thereby providing a lower background signal while preserving the
protein’s antigenic epitopes. And lastly, if the digested fragments
of DQ ovalbumin accumulate in organelles at a high concentration, the BODIPY FL fluorophore can potentially form excimers,
resulting in a shift of the fluorophore’s emission maximum from
515 nm (green) to ~620 nm (red). Although we offer the greenfluorescent DQ Green BSA and red-fluorescent DQ Red BSA
(D-12050, D-12051; Section 10.4), which are also self-quenched
BODIPY FL and BODIPY TR conjugates, we highly recommend
DQ ovalbumin (D-12053) for studying antigen processing and
presentation because ovalbumin is internalized via the mannose
receptor–mediated endocytosis pathway and is thus processed
more efficiently by antigen-presenting cells than is BSA.179
Antigen processing of DQ ovalbumin by both eosinophils 180
and immature dendritic cells 173 has been reported.
(E-12056), EnzChek Lysozyme Assay Kit (E-22013) — as well as the standalone quenched substrates DQ BSA (D-12050, D-12051), DQ collagen
(D-12052, D-12060), DQ ovalbumin (D-12053) and DQ gelatin (D-12054)
— relieves the intramolecular self-quenching, yielding brightly fluorescent
reaction products.
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
Fluorescent Casein
Using fluorescein-labeled casein (C-2990), researchers have
demonstrated casein-specific chemotaxis receptors in human
neutrophils and monocytes with flow cytometry.181 Neutrophils
activated with phorbol myristate acetate have been shown to
undergo a dose-dependent increase in binding of fluoresceinlabeled casein.182 It has also been demonstrated that fluoresceinlabeled casein reversibly binds to specific receptors on human
monocytes but does not bind to lymphocytes in vitro.181
EnzChek Protease Assay Kits
Our patented EnzChek Protease Assay Kits (E-6638, E-6639),
which provide convenient fluorescence-based assays for protease
activity, contain either green-fluorescent BODIPY FL casein or
red-fluorescent BODIPY TR-X casein.183 These casein-based
substrates are heavily labeled and therefore highly quenched conjugates; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Protease-catalyzed hydrolysis relieves this
quenching, yielding brightly fluorescent BODIPY FL– or BODIPY
TR-X–labeled peptides (Figure 16.15). In addition to their utility
for detecting protease contamination in culture medium and other
experimental samples, BODIPY FL casein and BODIPY TR-X
casein appear to have significant potential as nontoxic and pHinsensitive general markers for phagocytic cells in culture. In unpublished work, we have shown that uptake of these quenched
conjugates by neutrophils is accompanied by hydrolysis of the
labeled proteins by intracellular proteases and the generation of
fluorescent products that are well retained in cells. Once formed,
small BODIPY FL– or BODIPY TR-X–labeled peptides may
diffuse into other organelles or pass through gap junctions. This
phagocytosis assay is readily performed in a fluorescence microplate reader or a flow cytometer; localization of the fluorescent
products can be determined by fluorescence microscopy. Our
RediPlate 96 (R-22130, R-22132) and RediPlate 384 (R-22131,
R-22133) versions of these substrates (Section 10.4) are ideal for
high-throughput screening of potential protease inhibitors.
Alexa Fluor 488 Histones
The Alexa Fluor 488 conjugate of the lysine-rich calf thymus
histone H1 (H-13188) should be a useful probe for yeast nuclear
protein transport assays 184 and may be useful for studying nuclei
in live cells in experiments similar to those described in the literature.185,186 Unlike fluorescein conjugates of histones, Alexa Fluor
488 histones have pH-insensitive fluorescence and exceptional
photostability (Figure 1.11, Figure 1.42).
Probes for the Acrosome Reaction
Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity
of serine proteases and binds to acrosin, an acrosomal serine
protease associated with binding of spermatozoa to the zona
pellucida.187 Our Alexa Fluor 488 dye–labeled trypsin inhibitor
from soybean (T-23011) is useful for monitoring the surface
expression of acrosin during the acrosome reaction of spermatogenesis.188 The conjugate has been reported to have no effect on
sperm mobility, survival or zona binding capability, allowing
simultaneous assessment of acrosomal status and motility.188
A fluorescent peanut lectin has been combined with ethidium
homodimer-1 (EthD-1, E-1169; Section 15.2) for a combined
acrosome reaction assay and vital staining.189 Our Alexa Fluor
488 dye–labeled PNA (L-21409) may have similar utility as an
acrosomal stain. Wheat germ agglutinin (WGA) and Phaseolus
vulgaris lectin conjugates (Section 7.7) are also known to bind to
acrosomal structures of spermatids.190,191
BODIPY FL Hyaluronic Acid
Hyaluronic acid, a major glycosaminoglycan (GAG) constituent of the extracellular matrix, participates in the adhesion, motility, proliferation, and differentiation of cells, and is involved in
inflammation and cancer.192 The numerous biological effects of
this relatively simple, unbranched polysaccharide are exerted
through interactions with proteins, including the cell-surface
receptors CD44 and RHAMM (receptor for hyaluronic acid–
mediated motility).193,194 CD44, a 90,000-dalton transmembrane
adhesion glycoprotein, is frequently overexpressed by tumor
cells.195,196 A BODIPY FL dye–labeled hyaluronic acid derivative
similar to that described by Luo and Prestwich 197 is available
from Molecular Probes (H-23379). Receptor-mediated internalization of BODIPY FL dye–conjugated hyaluronic acid by human
ovarian cancer cells has been visualized by confocal laser-scanning microscopy,197 providing a foretaste of its anticipated broad
utility for direct detection of hyaluronic acid–protein interactions.
Oregon Green 488 Mucin
Mucins are large molecules composed of glycoproteins, sialic
acid and various sugars 198,199 that constitute a major component
of gastrointestinal, ocular, nasal and cervical mucosal tissues,
serving as a defensive barrier against bacterial invasion.200 Mucins are key elements in the understanding of several gastrointestinal and respiratory diseases, including cystic fibrosis and colon
cancer.201,202 Mucins bind to galectins — natural human lectins
found in some cancer cells 203 — and are internalized by at least
some cells.204 Our green-fluorescent Oregon Green 488 conjugate
of mucin from bovine submaxillary gland (M-23361) may assist
researchers in detection of mucin-binding cells by fluorescence
imaging and flow cytometry.
Fluorescent Cholera Toxin Subunit B — Markers of Lipid Rafts
Fluorescent cholera toxins (see Cholera Toxin Subunits A and B
in Section 7.7) are markers of lipid rafts — regions of cell membranes high in ganglioside GM1 that are thought to be important in
cell signaling.205,206 Cholera toxin subunit B (C-22851) is a powerful tool for the retrograde labeling of neurons (Section 14.3). Our
Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 and Alexa
Fluor 647 conjugates of the nontoxic B subunit of cholera toxin
(C-22841, C-22843, C-22842, C-22844) combine this versatile
tracer with the superior brightness of our Alexa Fluor dyes to provide sensitive and selective receptor labeling and neuronal tracing.
Fluorescent α-Crystallin
α-Crystallin is a large, polydisperse heat-shock protein that
accounts for up to 50% of the total protein content in vertebrate
lenses. This protein complex consists of many copies of two types
of homologous subunits, αA and αB crystallin, and has an average molecular mass of ~800,000 daltons.207 In the eye, α-crystallin helps to maintain the proper refractive index of the lens, which
it may do in part by acting as a molecular chaperone to prevent
denaturation of other lens proteins.207–209 Fluorescent α-crystallin
conjugates have been used to characterize the interaction of this
protein with fiber cell plasma membranes, which increases with
age or the onset of cataracts.210,211 To facilitate these investiga-
Section 16.1
681
tions, Molecular Probes offers a green-fluorescent conjugate of α-crystallin using our
outstanding Alexa Fluor 488 dye (C-23010), which yields bright, photostable and pHinsensitive fluorescence between pH 4 and pH 10 (Figure 1.11).
Figure 16.16 Live nerve terminals of motorneurons that innervate a rat lumbrical muscle stained
with the activity-dependent dye, FM 1-43 (T-3163)
and observed under low magnification. The dye
molecules, which insert into the outer leaflet of the
surface membrane, are captured in recycled synaptic vesicles of actively firing neurons. Image contributed by William J. Betz, Department of Physiology,
University of Colorado School of Medicine.
ProLong Antifade Kit
Photobleaching is the most consistently troublesome practical problem in
fluorescence microscopy, degrading the
signal-to-noise characteristics of images
and limiting the time available for data
acquisition. Addition of protective antifade reagents to the specimen is the
most straightforward practical measure
for counteracting photobleaching, as it
does not require compromises in exposure time, scan rate, magnification and
other imaging configuration parameters.
Among the numerous antifade reagents
that have been developed, Molecular
Probes’ ProLong reagent (see Section
24.1) provides the most consistent
performance in the widest range of
experimental setups. Outstanding features of the ProLong reagent include:
• Compatibility with a wide range of
different dyes and probes
• Uniform effectiveness — the ProLong
reagent does not distort the balance
of fluorescence signals in multicolor
analysis applications
• A turnkey solution to photobleaching
problems. No optimization is required
• Extended duration of the fluorescence
signal with minimal quenching of its
instantaneous intensity
• Formation of a dry mounting medium
allowing long-term preservation of
specimens with continued antifade
potency
682
Fluorescent Chemotactic Peptide
A variety of white blood cells containing the formyl-Met-Leu-Phe (fMLF) receptor
respond to bacterial N-formyl peptides by migrating to the site of bacterial invasion and
then initiating an activation pathway to control the spread of infection. Activation involves Ca2+ mobilization,212,213 transient acidification,214,215 actin polymerization,216
phagocytosis 217 and production of oxidative species.218 Molecular Probes offers the
fluorescein conjugate of the hexapeptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys (F-1314),
which has been extensively employed to investigate the fMLF receptor.219–223 This probe
is well suited for instruments that employ the argon-ion laser, including confocal laserscanning microscopes and flow cytometers. The fluorescein-labeled chemotactic peptide
has been used to study G-protein coupling and receptor structure,224,225 expression,226,227
distribution 228–230 and internalization.231 For studies of fMLF receptor internalization, the
fluorescence of extracellular labeled N-formyl hexapeptides may be quenched by adding
the anti-fluorescein/Oregon Green antibody (Section 7.4, Table 7.13). Alternatively,
reducing the pH of the external medium or adding trypan blue (Figure 16.23) can quench
extracellular fluorescence of the pH-sensitive fluorescein conjugate.
Fluorescent Insulin
Molecular Probes has prepared a high-purity, zinc-free fluorescein conjugate of human insulin (FITC insulin, I-13269). Unlike most commercially available preparations,
our FITC insulin is purified by HPLC and consists of a singly labeled species of insulin
that has been specifically modified at the N-terminus of the B-chain. Because the degree
and position of labeling can alter the biological activity of insulin, we have isolated the
singly labeled species that has been shown to retain its biological activity in an autophosphorylation assay.232 Our FITC insulin preparation should be useful for studying the
insulin receptor, as well as for conducting insulin-binding assays using fluorescence
polarization and flow cytometry techniques.
Fluorescein-Labeled α-MSH Analog
[Nle4, D-Phe7]-α-MSH is an analog of the tridecapeptide α-melanocyte-stimulating
hormone (α-MSH, melanotropin) with more potent and prolonged biological activity
than native α-MSH.233 α-MSH is active in fetal development, learning and memory
processes, and its receptors are important markers for the detection of melanomas. To
facilitate further investigation of these processes, we offer an N-terminal fluorescein–
labeled analog of [Nle4, D-Phe7]-α-MSH 234 (M-13440).
Fluorescent Dexamethasone Probe for Glucocorticoid Receptors
The synthetic steroid hormone dexamethasone binds to the glucocorticoid receptor,
producing a steroid–receptor complex that then localizes in the nucleus and regulates
gene transcription. In hepatoma tissue culture (HTC) cells, tetramethylrhodaminelabeled dexamethasone has been shown to have high affinity for the glucocorticoid
receptor in a cell-free system and to induce tyrosine aminotransferase (TAT) expression
in whole cells, albeit at a much lower rate than unmodified dexamethasone.235 This
labeled dexamethasone also allowed the first observations of the fluorescent steroid–
receptor complex in the HTC cell cytosol.235 Molecular Probes’ fluorescein dexamethasone (D-1383) should be similarly useful for studying the mechanism of glucocorticoid
receptor activation.
Methods for Detecting Internalized Fluorescent Ligands
Many of the fluorescent ligands described in this section first bind to cell-surface
receptors, then are internalized and, in some cases, recycled to the cell surface. Consequently, it can be difficult to assess whether the fluorescent signal is emanating from the
cell surface, the cell interior or, as is more typical, a combination of the two sites. Furthermore, the fluorophore’s sensitivity to environmental factors — principally intracellu-
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
lar pH — can affect the signal of the fluorescent ligand. Fortunately, there are means by
which these signals can be separated and, in some cases, quantitated. These include:
• Use of antibodies to the Alexa Fluor 488, BODIPY FL, fluorescein/Oregon Green,
tetramethylrhodamine, Texas Red and Cascade Blue dyes (Section 7.4, Table 7.13) to
quench most of the fluorescence of surface-bound or exocytosed probes. In addition,
these anti-fluorophore antibodies enable researchers to develop their cell or tissue
preparations for electron microscopy using standard horseradish peroxidase/diaminobenzidine 236 or colloidal gold 237 methodology or our NANOGOLD and Alexa
Fluor FluoroNanogold 1.4 nm gold cluster antibody and streptavidin conjugates
(Section 7.3, Section 7.6) followed by silver enhancement with the LI Silver (LIS)
Enhancement Kit (L-24919, Section 7.3, Figure 7.60).
• Use of a dye such as trypan blue to quench external fluorescent signals but not internalized signals 238,239 (Figure 16.23) — a method employed in our Vybrant Phagocytosis Assay Kit (V-6694).
• Rapid acidification of the medium to quench the fluorescence of pH-sensitive fluorophores such as fluorescein on the cell’s surface, thus enabling the selective detection
of the endocytosed probe.
• Tagging of proteins, polysaccharides, cells, bacteria, yeast, fungi 240 and other
materials to be endocytosed with a pH-sensitive dye — especially one of our SNARF,
SNAFL or Oregon Green dyes (Chapter 21) — that undergoes a spectral shift
(Figure 21.13, Figure 21.21) or intensity change in the acidic pH range found in
phagovacuoles and early endosomes.
• Use of heavily labeled proteins such as our DQ BSA and DQ gelatin probes that
undergo intracellular proteolysis to highly fluorescent peptides 241 (Section 10.4).
• Use of the optical sectioning capabilities of confocal laser-scanning microscopes, or
the multiphoton capabilities of certain microscopes, to make direct measurements of
internalized probes.
Membrane Markers of Endocytosis and Exocytosis
FM 1-43
Molecular Probes’ membrane probes FM 1-43, FM 2-10, FM 4-64 and FM 5-95 are
excellent reagents both for identifying actively firing neurons and for investigating the
mechanisms of activity-dependent vesicle recycling in widely different species.242–245
FM dyes may also be useful as general-purpose probes for investigating endocytosis and
for simply identifying cell membrane boundaries. These water-soluble dyes, which are
nontoxic to cells and virtually nonfluorescent in aqueous medium, are believed to insert
into the outer leaflet of the surface membrane where they become intensely fluorescent.
In a neuron that is actively releasing neurotransmitters, these dyes become internalized
within the recycled synaptic vesicles and the nerve terminals become brightly stained
(Figure 16.16, Figure 16.17). The nonspecific staining of cell-surface membranes can
simply be washed off prior to viewing. The amount of FM 1-43 taken up per vesicle by
endocytosis equals the amount of dye released upon exocytosis, indicating that the dye
does not transfer from internalized vesicles to an endosome-like compartment during the
recycling process.246 This feature permits quantitative fluorescence measurements to
be made. Like most styryl dyes, the absorption and fluorescence emission spectra of
FM 1-43 (absorption/emission maxima of 510/626 nm in methanol) are significantly
shifted in the membrane environment (Figure 14.61); FM 1-43 is efficiently excited with
standard fluorescein optical filters (Table 24.8) but poorly excited with standard tetramethylrhodamine optical filters.
Betz and his colleagues, who were the first to employ the styrylpyridinium dye
FM 1-43 (T-3163, Figure 16.18) to observe vesicle cycling in living nerve terminals,247–249 have used this important probe to examine the effects of okadaic acid on
synaptic vesicle clustering.250 FM 1-43 has been used to investigate synaptosomal recycling in a range of species including frog, rat and mouse.245,248,249,251–254 It has also been
shown that FM 1-43 can be used in Drosophila larvae, thereby facilitating the analysis of
mutations in synaptic vesicle recycling.255 FM 1-43 was employed in a study showing
that synaptosomal endocytosis is independent of both extracellular Ca2+ and membrane
Figure 16.17 A feline mesenteric Pacinian corpuscle labeled with the vital stain FM 1-43 (T-3163).
Image contributed by Michael Chua, University of
North Carolina at Chapel Hill.
Figure 16.18 T-3163 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide
(FM 1-43).
TECHNICAL
NOTE
Molecular Probes’
FM Dyes
The FM dyes from Molecular Probes
are water soluble and essentially nonfluorescent until bound to cell membranes.
The nontoxic dyes can be used to outline
cells and control for motion artifacts in
multicolor imaging experiments. They are
extensively used to follow synaptosome
recycling in neurons. FM dyes are available with distinguishable fluorescence
emissions. There are hundreds of references for these products at our Web site
(www.probes.com/search).
Section 16.1
683
potential in dissociated hippocampal neurons,253 as well as in a spectrofluorometric assay
demonstrating that nitric oxide–stimulated vesicle release is independent of Ca2+ in
isolated rat hippocampal nerve terminals.256 In addition, FM 1-43 has been used in combination with fura-2 (Section 20.2) to simultaneously measure intracellular Ca2+ and
membrane turnover.257 In an alternative approach, sulforhodamine 101 (S-359, Section
14.3), a red-fluorescent dye that is not taken up by synaptic vesicles, has been used to
reduce nonsynaptic fluorescence in FM 1-43 dye–labeled rat hippocampal slices.258 FM
1-43 dye–mediated photoconversion has been used to visualize recycling vesicles in
hippocampal neurons.259
Figure 16.19 Feline muscle spindle, a specialized
sensory receptor unit that detects muscle length
and changes in muscle length and velocity, was labeled with the vital stain FM 2-10 (T-7508). Image
contributed by Michael Chua, University of North
Carolina at Chapel Hill.
Figure 16.20 Correlated fluorescence imaging of
membrane migration, protein translocation and
chromosome localization during Bacillus subtilis
sporulation. Membranes were stained with redfluorescent FM 4-64 (T-3166, T-13320). Chromosomes were localized with the blue-fluorescent
nuclear counterstain, DAPI (D-1306, D-3571,
D-21490). The small, green-fluorescent patches
(top row) indicate the localization of a GFP fusion
to SpoIIIE, a protein essential for both initial membrane fusion and forespore engulfment. Progression of the engulfment is shown from left to right.
Green fluorescence in the middle and bottom rows
demonstrates fully engulfed sporangia stained with
MitoTracker Green FM (M-7514). Full details of the
experimental methods and interpretation are published in Proc Natl Acad Sci U S A 96, 14553
(1999). Image contributed by Kit Pogliano and
Marc Sharp, University of California at San Diego.
Reproduced from the 7 December 1999 issue of
Proc Natl Acad Sci U S A, with permission.
The 4-Di-1-ASP, 4-Di-2-ASP, TMADPH and TMAP-DPH probes are
essentially nonfluorescent until bound
to cell membranes or organelles.
684
Analogs of FM 1-43
A comparison of mammalian motor nerve terminals stained with either FM 1-43 or
the more hydrophilic analog FM 2-10 (T-7508, Figure 16.19) revealed that lower background staining by FM 2-10 and its faster destaining rate may make it the preferred
probe for quantitative applications.244,245 However, staining with FM 2-10 requires much
higher dye concentrations (100 µM compared with 2 µM for FM 1-43).245 FM 4-64
(T-3166, T-13320; Figure 14.62) and RH 414 (T-1111) — both more hydrophobic than
FM 1-43 — may also be useful as probes for investigating endocytosis. Because small
differences in the polarity of these probes can play a large role in their rates of uptake
and their retention properties, we have introduced FM 5-95 (T-23360), a slightly less
lipophilic analog of FM 4-64 with essentially identical spectroscopic properties. FM 4-64
exhibits long-wavelength red fluorescence that can be distinguished from the green
fluorescence of FM 1-43 with the proper optical filter sets (Table 24.8), thus permitting
two-color observation of membrane recycling in real time; 260,261 Membranes stained with
RH 414 typically exhibit orange fluorescence when observed through a longpass optical
filter that permits passage of light beyond 510 nm.248,249,262
Using time-lapse video fluorescence microscopy, Heuser and colleagues were able
to follow the internalization of FM 4-64 from the Dictyostelium plasma membrane into
the contractile vacuole and to observe up to 10 contractile vacuole cycles before the dye
redistributed to the endosomes.263 In addition, FM 4-64 staining has been used to visualize
membrane migration and fusion during Bacillus subtilis sporulation — movements that can
be correlated with the translocation of green fluorescent protein–labeled proteins 264 (Figure
14.62, Figure 16.20). FM 4-64 has also been reported to selectively stain yeast vacuolar
membranes and is proving to be an important tool for visualizing vacuolar organelle morphology and dynamics and for studying the endocytic pathway and vacuole fusion in
yeast 265–270 (Section 12.3). FM 4-64 is an endosomal marker and vital stain that persists
through cell division 271,272 and is a stain for functional presynaptic boutons.273
4-Di-1-ASP and 4-Di-2-ASP
Some cationic mitochondrial dyes such as 4-Di-1-ASP (D-288) and 4-Di-2-ASP
(D-289) stain presynaptic nerve terminals independent of neuronal activity.274–276 The
photostable 4-Di-2-ASP dye, which is nontoxic to cells, has been employed to stain
living nerve terminals in rabbit corneal epithelium,277 in rat epidermis 274 and at mouse,
snake and frog neuromuscular junctions,278–281 as well as to visualize the innervation of
the human choroid 282 and whole mounts of the gastrointestinal tract.283 4-Di-2-ASP
staining of neuromuscular junctions reportedly persists for several months in living
mice.281 Methods for using 4-Di-2-ASP to image neuronal cells in live animals have been
described 284 (Figure 16.21).
TMA-DPH and TMAP-DPH
Also useful as a lipid marker for endocytosis and exocytosis is the cationic linear
polyene TMA-DPH (T-204, Figure 13.42), which readily incorporates in the plasma
membrane of live cells.285,286 TMA-DPH is virtually nonfluorescent in water and is reported to bind to cells in proportion to the available membrane surface.287 Its fluorescence intensity is therefore sensitive to physiological processes that cause a net change
in membrane surface area, making it an excellent probe for monitoring events such as
changes in cell volume and exocytosis.287–290 During endocytosis, TMA-DPH progresses
from the cell periphery to perinuclear regions with little loss in fluorescence intensity.291
TMA-DPH can be extracted from plasma membranes by washing with medium, thus
providing a method for isolating cells that have only the internalized probe and monitor-
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
ing endocytosis.292–294 To provide supporting evidence for the maturation model for
endocytosis, researchers used TMA-DPH in fluorescence anisotropy–based assays to
investigate membrane fluidity in the plasma membrane and in successive endocytic
compartments of L929 cells.291 A protocol has been published that uses TMA-DPH for
labeling and flow-sorting of early endosomes.295
We also offer TMAP-DPH (P-3900), which has a three-carbon spacer between the
DPH fluorophore and the trimethylammonium substituent (Figure 16.22); the partitioning
properties of TMAP-DPH probe in multilamellar liposomes and use of the probe to assess
membrane fluidity have been described.296–299 Measurements of the fluorescence lifetime
of membrane-bound TMAP-DPH can be used to probe for hydration of lipid bilayers.300,301
Anti–Synapsin I Antibody
Synapsin I is an actin-binding protein that is localized exclusively to synaptic vesicles
and thus serves as an excellent marker for synapses in brain and other neuronal tissues.302,303 Synapsin I inhibits neurotransmitter release, an effect that is abolished upon
its phosphorylation by Ca2+/calmodulin–dependent protein kinase II (CaM kinase II).304
Antibodies directed against synapsin I have proven valuable in molecular and neurobiology
research, for example, to estimate synaptic density and to follow synaptogenesis.253,305–307
Molecular Probes offers anti–bovine synapsin I rabbit polyclonal antibody as an
affinity-purified IgG fraction (A-6442). This antibody was isolated from rabbits immunized against bovine brain synapsin I but is also active against human, rat and mouse
forms of the antigen; it has little or no activity against synapsin II. The affinity-purified
antibody was fractionated from the serum using column chromatography in which bovine
synapsin I was covalently bound to the column matrix. Affinity-purified anti–synapsin I
antibody is suitable for immunohistochemistry (Figure 18.12), Western blots, enzymelinked immunoadsorbent assays and immunoprecipitations.
Figure 16.21 Neuromasts, the external mechanosensory organs of fish, stained selectively with
the vital fluorescent probe 4-Di-2-ASP (D-289) in a
living larva of the Siamese fighting fish Betta
splendans at ten days postfertilization. In this dorsal view, staining was accomplished by putting live
fish into a dilute aqueous solution of the dye.
Image contributed by Paula Mabee, Department of
Biology, San Diego State University.
Figure 16.22 P-3900 N -((4-(6-phenyl-1,3,5hexatrienyl)phenyl)propyl)trimethylammonium
p-toluenesulfonate (TMAP-DPH).
High Molecular Weight Polar Markers
BioParticles Fluorescent Bacteria and Yeast
Molecular Probes’ BioParticles product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes and natural antigenicities. These fluorescent BioParticles products have been employed to study
phagocytosis by fluorescence microscopy,308 quantitative spectrofluorometry 309 and flow
cytometry.310–313 We offer Escherichia coli (K-12 strain), Staphylococcus aureus (Wood
strain without protein A) and zymosan (Saccharomyces cerevisiae) BioParticles covalently labeled with a variety of fluorophores, including fluorescein, Alexa Fluor 488,
BODIPY FL, tetramethylrhodamine and Texas Red dyes (Table 16.3). Special care has
been taken to remove any free dye after conjugation. Our Alexa Fluor 488 conjugate of
the E. coli BioParticles (E-13231) has exceptionally bright and photostable green fluorescence. Unlike the fluorescence of fluorescein-labeled BioParticles fluorescent bacteria
and yeast, which is partially quenched in acidic environments (Figure 21.2), fluorescence
of the Alexa Fluor 488, BODIPY FL, tetramethylrhodamine and Texas Red conjugates
is uniformly intense between pH 3 and 10. This property may be particularly useful in
quantitating fluorescent bacteria and zymosan within acidic phagocytic vacuoles. Our
The BioParticles fluorescent bacteria
and yeast are derivatives of natural
probes for modeling phagocytosis by
fluorescent microscopy, spectrofluorometry and flow cytometry. The Alexa
Fluor 488 and Alexa Fluor 594 conjugates are recommended as the best
green- and red-fluorescent analogs,
respectively, for phagocytosis studies.
Table 16.3 Molecular Probes’ selection of BioParticles fluorescent bacteria and yeast.
Label
(Abs/Em Maxima)
Escherichia coli
(K-12 strain)
Staphylococcus aureus
(Wood strain without protein A)
Zymosan A
(Saccharomyces cerevisiae)
Fluorescein (494/518)
E-2861
S-2851
Z-2841
Alexa Fluor 488 (495/519)
E-13231
S-23371
Z-23373
BODIPY FL (505/513)
E-2864
S-2854
Z-2844
S-23372
Z-23374
S-2859
Z-2849
Tetramethylrhodamine (555/580)
E-2862
Alexa Fluor 594 (590/617)
E-23370
Texas Red dye (595/615)
E-2863
Unlabeled
Z-2843
Section 16.1
685
BioParticles products, which are freeze-dried and ready for reconstitution in the buffer of choice, come with a general protocol
for measuring phagocytosis. We also offer opsonizing reagents
for use with each particle type as described below.
Fluorescent bacteria and yeast particles are proven tools for
studying a variety of parameters influencing phagocytosis; for
example, they have been used to:
• Detect the phagocytosis of yeast by murine peritoneal macrophage 314 and human neutrophils 309
• Determine the effects of different opsonization procedures on
the efficiency of phagocytosis of pathogenic bacteria 315 and
yeast 309
• Investigate the kinetics of phagocytosis degranulation and
actin polymerization in stimulated leukocytes 309
• Measure phagocytosis and, in conjunction with dihydroethidium (hydroethidine, D-1168, D-11347, D-23107;
Section 19.2), oxidative bursts in leukocytes using flow
cytometry 316,317
• Quantitate the effects of anti-inflammatory drugs on phagocytosis 309
• Show that Dictyostelium discoideum depleted of clathrin
heavy chains are still able to undergo phagocytosis of fluorescent zymosans 318
• Study molecular defects in phagocytic function 319
Trypan blue and other quenchers can quench the fluorescence of BioParticles that are bound to the surface but not
internalized 238,239,310 (Figure 16.23). Phagocytosis of viable
Candida albicans that were loaded with 5-(and-6)-carboxy
SNARF-1, acetoxymethyl ester, acetate (C-1271, C-1272; Section
21.2), a cell-permeant pH indicator, has been quantitated on a
single-cell level by flow cytometry without interfering cell autofluorescence.320 In addition to cellular applications, our fluorescent BioParticles products may be effective as flow cytometry
calibration references when sorting bacteria and yeast mutants.
These small particles may also be useful references for light
scattering studies because their sizes and shapes differ in characteristic ways.
Vybrant Phagocytosis Assay Kit
Our Vybrant Phagocytosis Assay Kit (V-6694) provides a
convenient set of reagents for quantitating phagocytosis and
assessing the effects of certain drugs or conditions on this cellular
process. In this assay, cells of interest are incubated first with
green-fluorescent E. coli BioParticles, which are internalized by
phagocytosis, and then with trypan blue, which quenches the
fluorescence of any extracellular BioParticles conjugate (Figure
16.23). The methodology provided by this kit was developed
using the adherent murine macrophage cell line J774; 239 however,
researchers can likely adapt this assay to other phagocytic cell
types. Each kit provides sufficient reagents for 250 tests using a
96-well microplate format and contains:
•
•
•
•
686
BioParticles fluorescein-labeled Escherichia coli
Hanks’ balanced salt solution (HBSS)
Trypan blue
Step-by-step instructions for performing the
phagocytosis assay
Opsonizing Reagents and Nonfluorescent BioParticles
Many researchers may want to use autologous serum to opsonize their fluorescent zymosan and bacterial particles; however,
we also offer special opsonizing reagents (E-2870, S-2860,
Z-2850) for enhancing the uptake of each type of particle, along
with a protocol for opsonization. These reagents are derived from
purified rabbit polyclonal IgG antibodies that are specific for the
E. coli, S. aureus or zymosan particles. Reconstitution of the
lyophilized opsonizing reagents requires only the addition of
water, and one unit of opsonizing reagent is sufficient to opsonize
~10 mg of the corresponding BioParticles product.
In addition, Molecular Probes offers nonfluorescent zymosan
(Z-2849) and S. aureus (S-2859) BioParticles. These nonfluorescent BioParticles are useful either as controls or for custom labeling with the reactive dye or indicator of interest.
Fluorescent Polystyrene Microspheres
Fluorescent polystyrene microspheres with diameters between
0.5 and 2.0 µm have been used to investigate phagocytic processes in rat and human neutrophils,321,322 human trabecular meshwork cells,323 mouse peritoneal macrophages,324,325 ciliated
protozoa 326,327 and Dictyostelium discoideum.328 The phagocytosis of fluorescent microspheres has been quantitated both with
image analysis 325 and with flow cytometry.324,326,327 Section 6.5
includes a detailed description of our full line of FluoSpheres
(Table 6.5) and TransFluoSpheres (Table 6.9) fluorescent microspheres. Because of their low nonspecific binding, carboxylatemodified microspheres appear to be best for applications involving phagocytosis. For phagocytosis experiments involving
multicolor detection, we particularly recommend our TransFluo-
Figure 16.23 Vybrant Phagocytosis Assay Kit (V-6694) for the simple
quantitation of phagocytosis. A) Briefly, phagocytic cells are incubated
with the green-fluorescent BioParticles (E-2861). B) The fluorescence
from any noninternalized BioParticles is then quenched by the addition of
trypan blue, and the samples are subsequently assayed with a fluorescence-based microplate reader equipped with filters for the detection of
fluorescein (FITC).
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
Spheres fluorescent microspheres, which have Stokes shifts up to 200 nm or more. Various opsonizing reagents such as rabbit serum or fetal calf serum have been used with the
microspheres to facilitate phagocytosis. Methods for coating fluorescent microspheres
with the CR1 complement receptor have also been described.329 Numerous references in
our extensive microspheres bibliography (M-8997) describe the use of fluorescent microspheres for phagocytosis studies.
Fluorescent Microspheres Coated with Collagen
Fibroblasts phagocytose and then intracellularly digest collagen. These activities play
an important role in the remodeling of the extracellular matrix during normal physiological turnover of connective tissues, in development, in wound repair and possibly in aging
and various disorders. A well-established procedure for observing collagen phagocytosis
by either flow cytometry or fluorescence microscopy involves the use of collagen-coated
fluorescent microspheres, which attach to the cell surface and become engulfed by fibroblasts.330–332 Molecular Probes offers yellow-green–fluorescent FluoSpheres collagen
I–labeled microspheres in either 1.0 µm or 2.0 µm diameters (F-20892, F-20893) for use
in these applications. In the production of these microspheres, collagen I from calf skin
is attached covalently to the microsphere’s surface.
Fluorescent Dextrans
Fluorescent dextrans can be used to monitor the uptake and internal processing of
exogenous materials by endocytosis.333–338 Molecular Probes offers dextrans with nominal molecular weights ranging from 3000 to 2,000,000 daltons, many of which can be
used as pinocytosis or phagocytosis markers (see Section 14.5 and Table 14.4 for further
discussion and a complete product list). Discrimination of internalized fluorescent dextrans from dextrans in the growth medium is facilitated by use of reagents that quench the
fluorescence of the external probe. For example, most of our anti-fluorophore antibodies
(Section 7.4, Table 7.13) strongly quench the fluorescence of the corresponding dyes. In
addition, although we are not aware of any publications that have used our Ca2+ indicator–conjugated dextrans as probes for uptake by cells, it should be possible to follow
endocytosis of dextran conjugates of fura-2, indo-1 and fluo-4 (Section 20.4) by using
Mn2+ to quench the fluorescence of the residual extracellular dextrans without affecting
intracellular dextrans. Using fluorescent dextran probes, researchers have investigated:
• Effects of the calmodulin antagonist W-13 on transcytosis and vesicular
morphology in Madin–Darby canine kidney (MDCK) cells 339
• Endocytic vesicles in Plasmodium falciparum 340
• Endocytosis-defective Chinese hamster ovary (CHO) cells 341 and Dictyostelium
discoideum 328,342–344
• Fusion competence of lysosomes in parasite-infected cells 345
• Kinetics of pinocytosis in mouse L cells by flow cytometry 346 and the suppression
of pinocytosis in mouse peritoneal macrophages by interferon 347
• Membrane changes in prostatic adenocarcinoma 348 and basophilic leukemia cells 349,350
• Methods to enhance receptor-mediated gene delivery 333
• Vacuolar morphology in Saccharomyces cerevisiae both during the cell cycle 351–353
and in mutant cell lines 354
A novel fluorometric assay employing dextrans to follow endosome fusion has been
described.355 Researchers incubated live baby hamster kidney (BHK) cells with a combination of a red-fluorescent dextran (Section 14.5) and BODIPY FL avidin and then
washed the cells to remove residual extracellular probe. This procedure pulse-labeled a
population of endosomes. Next, the cells were incubated with a biotinylated 10,000 MW
dextran, biotinylated transferrin or biotinylated bovine serum albumin to label a second
population of endosomes. Endosome fusion was then detected by the strong fluorescence
enhancement of the BODIPY FL dye that occurs when BODIPY FL avidin complexes
with biotinylated probes. Oregon Green 514 streptavidin (S-6369, Section 7.6) has a
fluorescence increase of more than 15-fold when it complexes with biotin; consequently,
we now recommend that probe for this application (Figure 16.24). Use of a red-fluorescent dextran as a fusion-insensitive reference enabled researchers to make ratiometric
measurements of the fused organelles. This method was later extended to study of the
Figure 16.24 Detection of endosomal fusion. A)
Cells are first incubated with a combination of a
high molecular weight, red-fluorescent dextran
(D-1830, D-1864, D-1829) and the green-fluorescent Oregon Green 514 streptavidin (S-6369),
which intrinsically has low fluorescence. B) The
cells are then incubated with a biotinylated probe
(e.g., biotinylated transferrin (T-23363)), and the
excess conjugate is washed. C) Endosomal fusion
is monitored by an increase in fluorescence by the
Oregon Green 514 dye as it is displaced by the biotinylated protein. The red-fluorescent dextran’s
fluorescence remains constant and allows for ratiometric measurements of the fused endosomes.
Section 16.1
687
cystic fibrosis transmembrane conductance regulator (CFTR) in
transfected Swiss 3T3 fibroblasts.356 Numerous other applications
of fluorescent dextran conjugates are described in our extensive
bibliography (D-8998) on dextrans.
pH Indicator Dextrans
The fluorescein dextrans (pKa ~6.4) are frequently used to
investigate endocytic acidification.337,357–361 Fluorescence of
fluorescein-labeled dextrans is strongly quenched upon acidification (Figure 21.2); however, fluorescein’s lack of a spectral shift
in acidic solution makes it difficult to discriminate between an
internalized probe that is quenched and residual fluorescence of
the external medium. Dextran conjugates that either shift their
emission spectra in acidic environments, such as the SNARF
dextrans (D-3303, D-3304; Section 21.4), or undergo significant
shifts of their excitation spectra, such as BCECF dextrans
(D-1878, D-1880; Section 21.4), are preferred. We also offer
dextrans labeled with the ratiometric, pH-sensitive, Oregon
Green 488 (pKa ~4.7) and Oregon Green 514 (pKa ~4.7) fluorophores. In addition to these pH indicator dextrans, Molecular
Probes prepares a dextran that is double-labeled with fluorescein
and tetramethylrhodamine (D-1950, D-1951; Section 21.4),
which has been used as a ratiometric indicator (Figure 21.33) to
measure endosomal acidification in Swiss 3T3 fibroblasts 362
and Hep G2 cells.363
Vitamin-Conjugated Probes
Low and colleagues have demonstrated that modification of
proteins by biotin,364,365 riboflavin 366 or folic acid 367–369 can
facilitate uptake of the labeled protein into at least some plant
cells (soybeans) and many mammalian cells, apparently via
receptor-mediated endocytosis. It has also been reported that
biotin covalently attached to cell surfaces induces existing receptors to endocytose avidin bioconjugates into nucleated cells (but
not into nonnucleated cells).370 Our biotin conjugates are listed in
Section 4.3, and our avidin and streptavidin conjugates in Table
7.17 of Section 7.6.
Low Molecular Weight Polar Markers
Hydrophilic fluorescent dyes — including sulforhodamine 101
(S-359), lucifer yellow CH (L-453), calcein (C-481), 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, pyranine; H-348) and
Cascade Blue hydrazide (C-687) — are thought to be taken up by
actively firing neurons through endocytosis-mediated recycling of
the synaptic vesicles.371,372 However, unlike the fluorescent FM
membrane probes described above, the hydrophilic fluorophores
appear to work for only a limited number of species in this application. Sulforhodamine 101 has been used to investigate neural
activity in turtle brainstem cerebellum,373 neonatal rat spinal
cord 374,375 and developing snake neuromuscular junctions.376
The ratiometric pH indicator (Figure 21.24) pyranine has been
employed to measure organelle acidification in the endosomal–
lysosomal pathway.377 The same dyes have frequently been used
as fluid-phase markers of pinocytosis.378–383 Pyranine has blue
fluorescence in acidic solutions and yellow fluorescence in neutral to basic solutions. Our highly water-soluble Alexa Fluor
hydrazides (Section 14.3, Table 3.1) provide superior spectral
properties and can be fixed in cells by aldehyde-based fixatives.
688
Carbohydrates
Fluorescent Glucose Analogs
Measurements of glucose uptake can be used to assess transport and viability in a variety of organisms. 2-NBD-deoxyglucose
(2-NBDG, N-13195) has been used to monitor glucose uptake in
pancreatic cells,384 the yeast Candida albicans 385 and the bacteria
Escherichia coli.386–388 Molecular Probes also offers the fluorescent glucose analog 6-NBD-deoxyglucose (6-NBDG, N-23106).
Using this probe, researchers have studied glucose uptake and
transport in isolated cells 389–391 and intact tissues.392 NBD fluorescence is environment sensitive, but typically displays excitation/emission maxima of ~465/540 nm and can be visualized
using optical filters designed for fluorescein (Table 24.8).
ManLev and ManLev Tetraacetate
The oligosaccharide components of cell-surface glycoproteins
play a role in the interactions that regulate many important biological processes, from cell–cell adhesion to signal transduction.
Because modification of these surface groups affects the behavior
of the cell,393 strategies that introduce alternative surface groups
to the cell 394 provide researchers with novel methods for tagging
specific cell populations.
Sialic acids are the most abundant terminal components of
oligosaccharides on mammalian cell-surface glycoproteins and
are synthesized from the six-carbon precursor N-acetylmannosamine.395 When cells in culture are incubated with 25 mM
N-levulinoyl-D-mannosamine 396 (ManLev, L-20492; Figure 3.9)
or — much more efficiently — with 25 µM ManLev tetraacetate
(L-20493), this ketone-containing monosaccharide serves as a
substrate in the oligosaccharide synthesis pathway, resulting in
ketone-tagged cell-surface oligosaccharides.397–402 And because
ketones are rare in cells, reaction with 2 mM biotinylated aldehyde-reactive probe (ARP, A-10550; Section 4.2; Figure 3.10)
followed by a fluorescent avidin or streptavidin conjugate (Section 7.6, Table 7.17) provides a means of identifying and tracing
tagged cells by either imaging (Figure 3.11) or flow cytometry.
We find ARP to be much more reactive than other biotin hydrazides and that fluorescent hydrazides usually are not suitable
for this detection because the concentrations that are required can
result in their internalization in live cells by pinocytosis.
Probes for Investigating Secretion
and Degranulation
Intracellular calcium plays an important role in stimulus–
secretion coupling, making Molecular Probes’ fluorescent Ca2+
indicators (see Chapter 20) among the commonly used tools for
investigating secretion. For example, researchers used our Fura
Red calcium indicator to show that Ca2+ spikes occurring in the
granular areas of rat pancreatic acinar cells could be correlated
with secretion.403 In addition, we offer probes that modify intracellular Ca2+ levels by a variety of mechanisms. For example,
thapsigargin (T-7458, Section 18.2), which inhibits Ca2+-ATPase–
mediated uptake of Ca2+ into the endoplasmic reticulum, has been
used to further define the role of intracellular Ca2+ in stimulus–
secretion coupling in a number of cell types.404–406
Fluorogenic and chromogenic substrates for enzymes that
are released during secretion or degranulation — such as
glucosaminidase, galactosidase, glucuronidase and acid phos-
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
phatase (see Chapter 10) — may also be useful for detecting
secretion. Many probes described in this and other chapters
have been used to detect, induce or block secretion or degranulation, including:
• BODIPY FL casein and BODIPY TR-X casein,183 substrates
in our EnzChek Protease Assay Kits (E-6638, E-6639), and
DQ Green BSA and DQ Red BSA (D-12050, D-12051),
which can detect secretion of proteases from cells, including
in agar plates,407 and may be useful for selecting enzymesecreting mutants
• Our EnzChek Elastase Assay Kit (E-12056, Section 10.4),
which may be useful for the continuous assay of elastase loss
from cells during secretion or degranulation 408
• Luciferin (L-2911, L-2912, L-2916; Section 10.5), which has
been used to detect ATP secretion from bovine adrenal chromaffin cells 409
• The Amplex Red Hydrogen Peroxide/Peroxidase Assay
Kit 410–412 (A-22188, Section 19.2), the OxyBURST Green
H2HFF BSA reagent (O-13291) and other reagents described
in Section 19.2 for detecting the production of extracellular
reactive oxygen species (ROS)
• TMA-DPH (T-204), which partially transfers from the plasma
membrane to the mast cell granule membrane during secretion, and acridine orange (A-1301, A-3568; Section 12.3),
which stains mast cell granules 289,413
• Anion-channel blockers such as DIDS and SITS (D-337,
A-339; Section 16.3), which block secretion 414–416
• Brefeldin A (B-7450, Section 12.4), which blocks transport of
NBD C6-ceramide and BODIPY FL C5-ceramide (N-1154,
N-22651, D-3521, B-22650; Section 12.4) through the Golgi
apparatus 417,418
• BODIPY FL C5-ceramide (D-3521, Section 12.4), which is
normally transported from the Golgi apparatus to the cell
surface and thus permits isolation of mutant mammalian cells
with defects in the secretory pathway 417
• Cyclic ADP-ribose (C-7619, Section 18.2), which induces
insulin secretion in pancreatic islets 419
• Nitric oxide donors, including SNAP (N-7892, N-7927;
Section 19.3), which have been shown to either inhibit 420
or stimulate 421 secretion
• Fluorescent analogs of polymyxin B (Section 18.3), an antibiotic that induces dose-dependent histamine and serotonin
release from mast cells 422–425
References
1. Traffic 1, 836 (2000); 2. Subcellular Biochemistry, Vol. 19: Endocytic Components: Identification
and Characterization, J.J.M. pp. 95–123 (1993);
3. J Immunol Methods 232, 23 (1999); 4. J Immunol 130, 1910 (1983); 5. J Leukoc Biol 43, 304
(1988); 6. J Immunol Methods 130, 223 (1990);
7. Biophys J 75, 2577 (1998); 8. J Leukoc Biol 64,
98 (1998); 9. J Biol Chem 270, 8328 (1995); 10. J
Biol Chem 269, 18535 (1994); 11. J Cell Physiol
156, 428 (1993); 12. Proc Natl Acad Sci U S A 74,
837 (1977); 13. J Cell Biol 121, 1257 (1993);
14. Bioconjug Chem 5, 105 (1994); 15. J Cell Sci
107, 2177 (1994); 16. Am J Pathol 138, 1173
(1991); 17. Biophys J 66, 1301 (1994); 18. Biophys J 67, 1280 (1994); 19. Methods Enzymol 98,
241 (1983); 20. In Vitro Cell Dev Biol 27A, 633
(1991); 21. J Lipid Res 34, 325 (1993); 22. J Biol
Chem 269, 20958 (1994); 23. Biol Reprod 50, 204
(1994); 24. Endocrinology 129, 3247 (1991);
25. Biol Reprod 47, 355 (1992); 26. J Cell Biol
101, 148 (1985); 27. Proc Natl Acad Sci U S A 81,
5454 (1984); 28. J Cell Biol 90, 595 (1981);
29. J Biol Chem 253, 9053 (1978); 30. J Supramol
Struct 13, 67 (1980); 31. J Cell Biol 82, 597
(1979); 32. Annu Rev Biochem 52, 223 (1983);
33. Arteriosclerosis 3, 2 (1983); 34. Arteriosclerosis 1, 177 (1981); 35. J Biol Chem 275, 9120
(2000); 36. J Biol Chem 265, 12722 (1990);
37. J Biol Chem 270, 1921 (1995); 38. J Biol
Chem 269, 21003 (1994); 39. J Cell Biol 129, 133
(1995); 40. Biochim Biophys Acta 1303, 193
(1996); 41. Eur J Cell Biol 60, 48 (1993); 42. J
Cell Biol 122, 923 (1993); 43. J Cell Biol 122, 417
(1993); 44. J Cell Biol 99, 2034 (1984); 45. J
Neurosci 6, 2163 (1986); 46. Exp Cell Res 198, 31
(1992); 47. J Biol Chem 267, 20383 (1992);
48. Histochem J 17, 1309 (1985); 49. J Neurosci
13, 4412 (1993); 50. J Neurosci 13, 4403 (1993);
51. J Exp Med 190, 523 (1999); 52. J Exp Med
190, 509 (1999); 53. J Biol Chem 271, 4100
(1996); 54. J Exp Med 181, 1743 (1995); 55. J
Exp Med 180, 1025 (1994); 56. J Exp Med 179,
269 (1994); 57. J Biol Chem 259, 10048 (1984);
58. J Exp Med 183, 1899 (1996); 59. Proc Natl
Acad Sci U S A 83, 5817 (1986); 60. Adv Immunol 28, 293 (1979); 61. Eur J Immunol 19, 125
(1989); 62. Blood 95, 198 (2000); 63. Circ Res 88,
491 (2001); 64. J Biol Chem 274, 34116 (1999);
65. Electron Microsc Rev 5, 381 (1992); 66. J
Periodontol 56, 553 (1985); 67. Biotechniques 28,
510 (2000); 68. Cell 61, 203 (1990); 69. J Biol
Chem 265, 7709 (1990); 70. Annu Rev Biochem
56, 881 (1987); 71. J Biol Chem 273, 28004
(1998); 72. EMBO J 5, 1181 (1986); 73. J Fluorescence 4, 295 (1994); 74. J Biol Chem 268, 23860
(1993); 75. J Membr Biol 118, 215 (1990);
76. Biochemistry 32, 12039 (1993); 77. J Cell Biol
109, 2105 (1989); 78. J Cell Biol 106, 1903
(1988); 79. Proc Natl Acad Sci U S A 75, 2135
(1978); 80. Mol Biol Cell 11, 747 (2000); 81. Mol
Biol Cell 9, 809 (1998); 82. J Immunol Methods
219, 57 (1998); 83. J Histochem Cytochem 40,
1353 (1992); 84. Cell 49, 423 (1987); 85. Trends
Biochem Sci 12, 350 (1987); 86. Biochimie 68,
375 (1986); 87. J Cell Biol 108, 1291 (1989);
88. Cell 37, 789 (1984); 89. Blood 95, 721 (2000);
90. Scand J Immunol 53, 393 (2001); 91. J Biol
Chem 276, 9649 (2001); 92. J Biol Chem 274,
22303 (1999); 93. J Biol Chem 274, 26233 (1999);
94. Mol Biol Cell 10, 3835 (1999); 95. Cancer Res
61, 7763 (2001); 96. J Biol Chem 276, 13096
(2001); 97. Mol Biol Cell 10, 4403 (1999); 98. J
Cell Biol 125, 253 (1994); 99. J Cell Biol 121, 61
(1993); 100. J Biol Chem 263, 8844 (1988);
101. J Biol Chem 275, 15271 (2000); 102. J Biol
Chem 275, 29636 (2000); 103. Biochemistry 31,
5820 (1992); 104. J Bioenerg Biomembr 23, 147
(1991); 105. J Biol Chem 266, 3469 (1991);
106. J Biol Chem 265, 6688 (1990); 107. Nature
358, 337 (1992); 108. J Cell Biol 123, 1119
(1993); 109. J Cell Biol 122, 1231 (1993); 110. J
Cell Biol 122, 565 (1993); 111. J Cell Biol 118,
267 (1992); 112. J Cell Biol 122, 1207 (1993);
113. J Cell Biol 125, 1265 (1994); 114. Nature
346, 335 (1990); 115. Eur J Cell Biol 79, 394
(2000); 116. J Biol Chem 272, 13929 (1997);
117. J Cell Biol 135, 1749 (1996); 118. Mol Biol
Cell 7, 355 (1996); 119. Pharmacol Toxicol 71, 3
(1992); 120. Nature 391, 499 (1998); 121. J
Immunol 139, 1647 (1987); 122. Biochemistry 36,
8359 (1997); 123. Scand J Immunol 46, 609
(1997); 124. Int J Biochem 26, 201 (1994);
125. Adv Exp Med Biol 443, 123 (1998);
126. Trends Microbiol 4, 185 (1996); 127. Microb
Pathog 14, 343 (1993); 128. J Bacteriol 181, 4417
(1999); 129. Infect Immun 65, 514 (1997);
130. Adv Exp Med Biol 443, 113 (1998);
131. Brain Res Brain Res Rev 27, 257 (1998);
132. Biol Reprod 43, 712 (1990); 133. FEMS
Microbiol Lett 176, 91 (1999); 134. Proc Natl
Acad Sci U S A 92, 9603 (1995); 135. Neurology
49, 717 (1997); 136. Lancet 347, 1614 (1996);
137. Eur J Biochem 213, 1205 (1993); 138. Eur J
Cell Biol 72, 247 (1997); 139. Parasitol Res 80,
403 (1994); 140. J Immunol 167, 2921 (2001);
141. J Biol Chem 270, 28812 (1995); 142. Thromb
Res 77, 543 (1995); 143. Biochem J 270, 149
(1990); 144. Biochem Pharmacol 36, 4035 (1987);
145. Biochemistry 33, 266 (1994); 146. J Biol
Chem 270, 11358 (1995); 147. J Cell Biol 104,
1403 (1987); 148. Eur J Med Res 3, 465 (1998);
149. J Biol Chem 258, 12582 (1983); 150. Science
231, 1559 (1986); 151. Proc Natl Acad Sci U S A
82, 8057 (1985); 152. J Cell Biol 147, 1419
(1999); 153. Br J Haematol 93, 204 (1996); 154. J
Lab Clin Med 123, 728 (1994); 155. Cytometry
17, 287 (1994); 156. Ann N Y Acad Sci 416, 426
Section 16.1
689
References — continued
(1983); 157. Blood 87, 602 (1996); 158. Blood 94,
3829 (1999); 159. FASEB J 15, 1044 (2001);
160. Proc Natl Acad Sci U S A 98, 12509 (2001);
161. Mol Cell 7, 811 (2001); 162. J Biol Chem
276, 25190 (2001); 163. Biochemistry 28, 1048
(1989); 164. J Cell Sci 101, 873 (1992); 165. Arch
Biochem Biophys 227, 358 (1983); 166. Biophys J
71, 2319 (1996); 167. J Cell Biol 87, 14 (1980);
168. Biochemistry 32, 8168 (1993); 169. Immunology, 3rd Ed., Roitt IM, Brostoff J, Male DK pp.
6–14 (1993); 170. Eur J Immunol 25, 1823 (1995);
171. J Immunol 149, 2894 (1992); 172. J Immunol
145, 417 (1990); 173. Proc Natl Acad Sci U S A
96, 15056 (1999); 174. Biol Cell 87, 95 (1996);
175. J Microsc 185, 339 (1997); 176. Biol Cell 90,
169 (1998); 177. Cytometry 28, 25 (1997); 178. J
Immunol 159, 2177 (1997); 179. Exp Cell Res
215, 17 (1994); 180. J Immunol 167, 3146 (2001);
181. Inflammation 7, 363 (1983); 182. J Immunol
139, 3028 (1987); 183. Anal Biochem 251, 144
(1997); 184. J Cell Biol 123, 785 (1993); 185. J
Cell Biol 109, 505 (1989); 186. Biotechniques 17,
730 (1994); 187. Mol Reprod Dev 53, 350 (1999);
188. Zygote 8, 127 (2000); 189. J Androl 19, 542
(1998); 190. Mol Reprod Dev 40, 164 (1995);
191. Res Vet Sci 69, 113 (2000); 192. Curr Opin
Cell Biol 12, 581 (2000); 193. Proteins 39, 103
(2000); 194. Trends Mol Med 7, 213 (2001);
195. Bioconjug Chem 10, 755 (1999); 196. Clin
Cancer Res 1, 333 (1995); 197. Bioconjug Chem
12, 1085 (2001); 198. Eur J Biochem 32, 148
(1973); 199. Biochim Biophys Acta 63, 235
(1962); 200. J Biol Chem 274, 31751 (1999);
201. Glycoconj J 15, 823 (1998); 202. Int J Cancer
82, 52 (1999); 203. Cancer Res 56, 4354 (1996);
204. J Infect Dis 158, 398 (1988); 205. Mol Biol
Cell 11, 1645 (2000); 206. Mol Microbiol 13, 745
(1994); 207. J Biol Chem 271, 29060 (1996);
208. Eye 13, 403 (1999); 209. Proc Natl Acad Sci
U S A 97, 3260 (2000); 210. Biochemistry 39,
15791 (2000); 211. J Biol Chem 275, 6664 (2000);
212. J Leukoc Biol 49, 369 (1991); 213. Trends
Biochem Sci 15, 69 (1990); 214. J Leukoc Biol
53, 673 (1993); 215. Biochem Biophys Res
Commun 122, 755 (1984); 216. Physiol Rev 67,
285 (1987); 217. J Cell Biol 82, 517 (1979);
218. J Biol Chem 265, 13449 (1990); 219. Biochemistry 29, 313 (1990); 220. J Biol Chem 265,
16725 (1990); 221. J Cell Biol 109, 1133 (1989);
222. Proc Natl Acad Sci U S A 78, 7540 (1981);
223. Science 205, 1412 (1979); 224. J Biol Chem
270, 10686 (1995); 225. J Biol Chem 269, 326
(1994); 226. J Immunol Methods 149, 159 (1992);
227. Biochemistry 29, 11123 (1990); 228. J Cell
Biol 121, 1281 (1993); 229. J Cell Sci 100, 473
(1991); 230. Biochem Soc Trans 18, 219 (1990);
231. J Biol Chem 259, 5661 (1984); 232. Anal
Chem 69, 4994 (1997); 233. Proc Natl Acad Sci
U S A 77, 5754 (1980); 234. J Pharm Sci 74, 237
(1985); 235. J Steroid Biochem 23, 267 (1985);
236. Histochemistry 84, 333 (1986); 237. J Cell
Biol 111, 249 (1990); 238. J Insect Physiol 40,
1045 (1994); 239. J Immunol Methods 162, 1
(1993); 240. Infect Immun 67, 885 (1999); 241. Eur
J Cell Biol 75, 192 (1998); 242. J Biol Chem 275,
15279 (2000); 243. J Neurochem 73, 2227 (1999);
244. Annu Rev Neurosci 22, 1 (1999); 245. Proc R
Soc Lond B Biol Sci 255, 61 (1994); 246. Nature
392, 497 (1998); 247. Annu Rev Physiol 60, 347
(1998); 248. J Neurosci 12, 363 (1992);
690
249. Science 255, 200 (1992); 250. J Cell Biol
124, 843 (1994); 251. Neuron 14, 983 (1995);
252. Neuron 14, 773 (1995); 253. Neuron 11,
713 (1993); 254. J Physiol 460, 287 (1993);
255. Neuron 13, 363 (1994); 256. Neuron 12,
1235 (1994); 257. Cell Calcium 18, 440 (1995);
258. Neuron 24, 803 (1999); 259. Proc Natl Acad
Sci U S A 98, 12748 (2001); 260. J Cell Biol 131,
679 (1995); 261. J Neurosci 15, 8246 (1995);
262. J Neurosci 15, 6327 (1995); 263. J Cell Biol
121, 1311 (1993); 264. Proc Natl Acad Sci U S A
96, 14553 (1999); 265. J Cell Biol 149, 397
(2000); 266. J Cell Biol 146, 85 (1999);
267. Science 285, 1084 (1999); 268. Proc Natl
Acad Sci U S A 94, 5582 (1997); 269. Proc Natl
Acad Sci U S A 94, 5662 (1997); 270. J Cell Biol
128, 779 (1995); 271. Science 282, 1516 (1998);
272. J Cell Biol 141, 625 (1998); 273. Neuron 17,
91 (1996); 274. Cell Tissue Res 255, 125 (1989);
275. J Neurosci 7, 1207 (1987); 276. Nature 310,
53 (1984); 277. J Neurosci 13, 4511 (1993);
278. J Neurosci 14, 5672 (1994); 279. J Neurosci
14, 3319 (1994); 280. J Neurosci 14, 796 (1994);
281. J Neurosci 7, 1215 (1987); 282. Ophthalmic
Res 26, 290 (1994); 283. J Auton Nerv Syst 38, 77
(1992); 284. Trends Neurosci 10, 398 (1987);
285. J Cell Biol 127, 725 (1994); 286. Cell Biophys 5, 129 (1983); 287. Biochemistry 25, 2149
(1986); 288. Biol Cell 79, 265 (1993); 289. Biochim Biophys Acta 1067, 71 (1991); 290. Biochim
Biophys Acta 901, 138 (1987); 291. J Cell Biol
125, 783 (1994); 292. Biol Cell 71, 293 (1991);
293. Biochim Biophys Acta 1030, 73 (1990);
294. Cell Biophys 14, 17 (1989); 295. Cell Biology: A Laboratory Handbook, 2nd Ed., Vol. 2, Celis
JE, Ed. pp. 63–69 (1998); 296. Mol Cell Biochem
206, 33 (2000); 297. Ann N Y Acad Sci 786, 274
(1996); 298. Chem Phys Lipids 66, 135 (1993);
299. J Fluorescence 3, 145 (1993); 300. Biophys J
72, 1264 (1997); 301. Biochemistry 36, 10630
(1997); 302. Science 226, 1209 (1984); 303. J Cell
Biol 96, 1337 (1983); 304. Molecular and Cellular
Mechanisms of Neurotransmitter Release, Stjarne
L, et al., Eds. pp. 31–45 (1994); 305. J Neurosci
12, 1736 (1992); 306. J Neurosci 11, 1617 (1991);
307. J Neurosci 9, 2151 (1989); 308. J Immunol
Methods 142, 31 (1991); 309. J Immunol Methods
112, 99 (1988); 310. Cytometry 17, 173 (1994);
311. J Clin Microbiol 30, 2071 (1992); 312. J
Immunol Methods 121, 203 (1989); 313. J Immunol 133, 3303 (1984); 314. J Immunol Methods
123, 259 (1989); 315. J Immunol Methods 116,
235 (1989); 316. J Immunol Methods 170, 117
(1994); 317. Cytometry 12, 687 (1991); 318. J
Cell Biol 118, 1371 (1992); 319. Diagn Clin
Immunol 5, 62 (1987); 320. Am J Physiol 267,
L211 (1994); 321. J Immunol Methods 88, 175
(1986); 322. Biochem J 266, 669 (1990);
323. Invest Ophthalmol Vis Sci 30, 2499 (1989);
324. Cell Immunol 156, 508 (1994); 325. J Leukoc
Biol 48, 403 (1990); 326. Cytometry 13, 423
(1992); 327. Cytometry 11, 875 (1990); 328. J
Cell Biol 126, 1433 (1994); 329. Cytometry 12,
677 (1991); 330. Exp Cell Res 237, 383 (1997);
331. J Cell Physiol 155, 461 (1993); 332. J Cell
Biol 98, 1947 (1984); 333. J Biol Chem 269,
12918 (1994); 334. Am J Physiol 258, C309
(1990); 335. J Cell Physiol 131, 200 (1987);
336. Proc Natl Acad Sci U S A 82, 8523 (1985);
337. Exp Cell Res 150, 36 (1984); 338. Proc Natl
Acad Sci U S A 80, 3334 (1983); 339. J Biol
Chem 269, 19005 (1994); 340. J Cell Biol 101,
2302 (1985); 341. J Biol Chem 268, 25357 (1993);
342. J Cell Biol 127, 387 (1994); 343. J Cell Biol
126, 343 (1994); 344. J Cell Biol 118, 1271
(1992); 345. Science 249, 641 (1990); 346. Eur J
Biol 34, 96 (1984); 347. J Cell Biol 98, 1328
(1984); 348. Cytometry 14, 826 (1993); 349. J
Cell Biol 101, 2156 (1985); 350. J Cell Biol 101,
2145 (1985); 351. J Cell Biol 107, 115 (1988);
352. Science 241, 589 (1988); 353. J Cell Biol
105, 1539 (1987); 354. J Cell Biol 107, 1369
(1988); 355. Biophys J 69, 716 (1995); 356. Proc
Natl Acad Sci U S A 93, 12484 (1996);
357. FASEB J 8, 573 (1994); 358. Proc Natl Acad
Sci U S A 91, 4811 (1994); 359. J Biol Chem 268,
6742 (1993); 360. J Cell Sci 105, 861 (1993);
361. Exp Cell Res 150, 29 (1984); 362. J Cell Biol
119, 99 (1992); 363. J Cell Biol 130, 821 (1995);
364. Plant Physiol 93, 1492 (1990); 365. Plant
Physiol 98, 673 (1992); 366. Biochim Biophys
Acta 1426, 195 (1999); 367. Biochim Biophys
Acta 1312, 237 (1996); 368. Proc Natl Acad Sci
U S A 88, 5572 (1991); 369. J Cell Sci 106, 423
(1993); 370. Bioconjug Chem 10, 1044 (1999);
371. Nature 314, 357 (1985); 372. Science 225,
851 (1984); 373. J Neurosci 12, 3187 (1992);
374. J Comp Neurol 396, 521 (1998); 375. J
Physiol 478, 265 (1994); 376. Neuron 11, 801
(1993); 377. Proc Natl Acad Sci U S A 92, 3156
(1995); 378. Cell Biology: A Laboratory Handbook, 2nd Ed., Vol. 2, Celis JE, Ed. pp. 495–500
(1998); 379. Am J Physiol 275, R697 (1998);
380. Microvasc Res 39, 1 (1990); 381. Am J Vet
Res 49, 663 (1988); 382. J Membr Biol 93, 237
(1986); 383. Proc Natl Acad Sci U S A 84, 1921
(1987); 384. J Biol Chem 275, 22278 (2000);
385. Appl Microbiol Biotechnol 46, 400 (1996);
386. J Microbiol Methods 42, 87 (2000);
387. Biochim Biophys Acta 1289, 5 (1996);
388. Biosci Biotechnol Biochem 60, 1899 (1996);
389. Cytometry 27, 262 (1997); 390. Biochemistry
34, 15395 (1995); 391. Biochim Biophys Acta
1111, 231 (1992); 392. Histochem J 26, 207
(1994); 393. Cell 1, 147 (1974); 394. Biotechniques 31, 384 (2001); 395. Glycobiology 1, 187
(1991); 396. US 6,075,134; 397. Nat Biotechnol
19, 553 (2001); 398. Methods Enzymol 327, 260
(2000); 399. Glycobiology 11, 11R (2001);
400. Glycobiology 10, 1049 (2000); 401. Science
276, 1125 (1997); 402. J Biol Chem 273, 31168
(1998); 403. EMBO J 12, 3017 (1993);
404. Biochim Biophys Acta 1220, 199 (1994);
405. J Neurochem 59, 2224 (1992); 406. Cell
Regul 2, 27 (1991); 407. Biophys J 74, 90 (1998);
408. J Leukoc Biol 41, 8 (1987); 409. J Neurochem 49, 1266 (1987); 410. Anal Biochem 253,
162 (1997); 411. J Clin Microbiol 29, 1836
(1991); 412. FASEB J 13, 69 (1999); 413. J
Histochem Cytochem 31, 737 (1983); 414. Endocrinology 125, 1231 (1989); 415. Exp Cell Res
140, 15 (1982); 416. Proc Natl Acad Sci U S A 77,
2721 (1980); 417. Mol Biol Cell 6, 135 (1995);
418. J Biol Chem 268, 2341 (1993); 419. Science
259, 370 (1993); 420. Biochem Biophys Res
Commun 195, 1354 (1993); 421. Am J Physiol
265, G418 (1993); 422. J Neurosci Methods 75,
103 (1997); 423. Anal Chem 68, 2897 (1996);
424. Agents Actions 14, 379 (1984); 425. Cell
Biophys 4, 245 (1982).
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
www.probes.com
Data Table — 16.1 Probes for Following Receptor Binding, Endocytosis and Exocytosis
Cat #
C-481
C-687
D-289
D-288
D-1383
D-2935
E-3476
E-3477
E-3478
E-3480
E-3481
E-7498
E-13345
F-1314
F-2902
H-348
L-453
L-3482
L-3483
L-3484
L-3485
L-20492
L-20493
L-23380
M-13440
N-13195
N-23106
P-3900
S-359
T-204
T-1111
T-3163
T-3166
T-7508
T-13320
T-23360
MW
622.54
596.44
394.30
366.24
840.98
584.37
~6100
~6600
~6500
see Notes
~6800
~6600
see Notes
1213.41
see Notes
524.37
457.24
see Notes
see Notes
see Notes
see Notes
277.27
445.42
see Notes
1963.13
342.26
342.26
503.70
606.71
461.62
581.48
611.55
607.51
555.44
607.51
565.43
Storage
L
L
L
L
L
F,D,AA
FF,D
FF,D
FF,D,L
FF,D,L
FF,D,L
FF,D,L
FF,D,L
F,L
RR,L,AA
D,L
L
RR,L,AA
RR,L,AA
RR,L,AA
RR,L,AA
F,DD
F,DD
RR,L,AA
F,D,L
F,L
F,L
D,L
L
D,L
D,L
D,L
D,L
D,L
D,L
D,L
Soluble
pH >5
H2O
H2O, DMF
DMF
pH >6, DMF
DMF
H2O
H2O
H2O
H2O
H2O
H2O
H2O
pH >6, DMF
H2O
H2O
H2O
see Notes
see Notes
see Notes
see Notes
H2O
DMSO
see Notes
H2O
H2O
H2O
DMF, DMSO
H2O
DMF, DMSO
DMSO, EtOH
H2O, DMSO
H2O, DMSO
H2O, DMSO
H2O, DMSO
H2O, DMSO
Abs
494
399
488
475
494
258
<300
<300
495
596
555
511
497
494
<300
454
428
554
515
554
510
<300
<300
495
494
466
467
354
586
355
532
510
558
506
558
560
EC
76,000
30,000
48,000
45,000
76,000
11,000
84,000
ND
85,000
85,000
ND
72,000
24,000
12,000
ND
ND
ND
ND
ND
78,000
20,000
22,000
85,000
108,000
75,000
55,000
56,000
46,000
50,000
46,000
43,000
Em
517
421
607
605
519
none
none
none
517
612
581
528
520
517
none
511
536
571
520
571
518
none
none
519
524
540
540
429
605
430
716
626
734
620
734
734
Solvent
pH 9
H2O
MeOH
MeOH
pH 9
MeOH
pH 8
pH 7
pH 7
pH 9
pH 8
pH 9
pH 9
H2O
see Notes
see Notes
see Notes
see Notes
see Notes
pH 9
MeOH
MeOH
MeOH
H2O
MeOH
MeOH
MeOH
CHCl3
MeOH
CHCl3
CHCl3
Notes
1
2, 3
4
5
6
6, 7
8, 9
6, 7
6, 7
8, 10
11, 12, 13
14
15
8, 16, 17, 18
8, 16, 17, 18
8, 16, 17, 18
8, 16, 17, 18
11
8,16,17,18
19
20
21
21
4, 22
4, 23
24
4
24
24
For definitions of the contents of this data table, see “How to Use This Book” on page viii.
Notes
1. C-481 fluorescence is strongly quenched by micromolar concentrations of Fe3+, Co2+, Ni2+ and Cu2+ at pH 7 (Am J Physiol 268, C1354 (1995); J Biol Chem 274, 13375 (1999)).
2. Cascade Blue dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.
3. Maximum solubility in water is ~1% for C-687.
4. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and
80 nm for emission, but varies considerably from one dye to another.
5. D-2935 is colorless and nonfluorescent until both the acetates are hydrolyzed and the product is subsequently oxidized. The product contains less than 0.1% of oxidized derivative
when initially prepared. The oxidation product is a 2′,7′-dichlorofluorescein derivative with spectra similar to C-368 (Section 14.3).
6. EGF conjugates have approximately one label per peptide.
7. The value of EC listed for this EGF conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide lableing ratio and no change of EC
due to dye–peptide interactions.
8. ND = not determined.
9. E-3480 is a complex of E-3477 with Texas Red streptavidin (S-872, Section 7.6), which typically incorporates three dyes/streptavidin (MW ~52,800).
10. E-13345 is a complex of E-3477 with Alexa Fluor 488 streptavidin (S-11223, Section 7.6), which typically incorporates four dyes/streptavidin (MW ~52,800).
11. This product is supplied as a ready-made solution in the solvent indicated under Soluble.
12. F-2902 is essentially colorless and nonfluorescent until oxidized. A small amount (~5%) of oxidized material is normal and acceptable for the product as supplied. The oxidation
product is fluorescent (Abs = 495 nm, Em = 524 nm) (J Immunol Methods 130, 223 (1990)).
13. This product consists of a dye–bovine serum albumin conjugate (MW ~66,000) complexed with IgG in a ratio of approximately 1:4 mol:mol (BSA:IgG).
14. H-348 spectra are pH dependent.
15. Maximum solubility in water is ~8% for L-453.
16. LDL complexes must be stored refrigerated BUT NOT FROZEN. The maximum shelf life under the indicated storage conditions is four to six weeks.
17. This LDL complex incorporates multiple fluorescent labels. The number of dyes per apoprotein B (MW ~500,000) is indicated on the product label.
18. LDL complexes are packaged under argon in 10 mM Tris, 150 mM NaCl, 0.3 mM EDTA, pH 8.3 containing 2 mM azide. Spectral data reported are measured in this buffer.
19. The value of EC listed for this peptide conjugate is that of the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change
of EC due to dye–peptide interactions.
20. NBD derivatives are almost nonfluorescent in water. QY and τ increase and Em decreases in aprotic solvents and other nonpolar environments relative to water (Photochem Photobiol
54, 361 (1991); Biochemistry 16, 5150 (1977)).
21. Diphenylhexatriene (DPH) and its derivatives are essentially nonfluorescent in water. Absorption and emission spectra have multiple peaks. The wavelength, resolution and relative
intensity of these peaks are environment dependent. Abs and Em values are for the most intense peak in the solvent specified.
22. RH 414 Abs ~500 nm, Em ~635 nm when bound to phospholipid bilayer membranes. Effectively nonfluorescent in H2O.
23. FM 1-43 Abs = 479 nm, Em = 598 nm bound to phospholipid bilayer membranes. Em = 565 nm bound to synaptosomal membranes (Neuron 12, 1235 (1994)). Effectively nonfluorescent in H2O.
24. FM 4-64 and FM 5-95 are nonfluorescent in water. Excitation at 515 nm and emission detection at 640 nm is a suitable configuration for imaging membrane staining with these dyes
(J Cell Biol 128, 779 (1995)).
Section 16.1
691
Product List — 16.1 Probes for Following Receptor Binding, Endocytosis and Exocytosis
Cat #
Product Name
A-6442
A-11130
C-481
C-687
C-2990
C-22841
C-22843
C-22842
C-22844
C-22851
C-13185
C-23010
D-1383
D-2935
D-289
D-288
D-12050
D-12053
D-12051
E-23377
E-6638
E-6639
E-3476
E-3477
E-13345
E-3480
E-3478
E-7498
E-3481
E-2870
E-13231
E-23370
E-2864
E-2861
E-2862
E-2863
F-2902
F-13191
F-13192
F-13193
F-7496
F-20892
F-20893
F-1314
G-13186
G-13187
H-13188
H-23379
H-348
I-13269
L-13350
L-21409
L-20492
L-20493
L-23351
L-23352
L-23353
L-23350
L-23356
L-23357
L-23358
L-23355
L-3486
L-23380
L-3485
anti-synapsin I (bovine), rabbit IgG fraction *affinity purified* ............................................................................................................................
anti-transferrin receptor (human), mouse IgG1, monoclonal 236-15375 ..............................................................................................................
calcein *high purity* ............................................................................................................................................................................................
Cascade Blue® hydrazide, trisodium salt ..............................................................................................................................................................
casein, fluorescein conjugate ...............................................................................................................................................................................
cholera toxin subunit B, Alexa Fluor® 488 conjugate ............................................................................................................................................
cholera toxin subunit B, Alexa Fluor® 555 conjugate ............................................................................................................................................
cholera toxin subunit B, Alexa Fluor® 594 conjugate ............................................................................................................................................
cholera toxin subunit B, Alexa Fluor® 647 conjugate ............................................................................................................................................
cholera toxin subunit B (recombinant) .................................................................................................................................................................
collagen, type IV from human placenta, Oregon Green® 488 conjugate ...............................................................................................................
α-crystallin from bovine eye lens, Alexa Fluor® 488 conjugate ............................................................................................................................
dexamethasone fluorescein ..................................................................................................................................................................................
2′,7′-dichlorodihydrofluorescein diacetate, succinimidyl ester (OxyBURST® Green H2DCFDA, SE) .....................................................................
4-(4-(diethylamino)styryl)-N-methylpyridinium iodide (4-Di-2-ASP) ...................................................................................................................
4-(4-(dimethylamino)styryl) -N-methylpyridinium iodide (4-Di-1-ASP) ...............................................................................................................
DQ™ Green BSA *special packaging* ..................................................................................................................................................................
DQ™ ovalbumin *special packaging* ...................................................................................................................................................................
DQ™ Red BSA *special packaging* .....................................................................................................................................................................
Endostatin™ protein (human, recombinant) *1 mg/mL* ......................................................................................................................................
EnzChek® Protease Assay Kit *green fluorescence* *100-1000 assays* ............................................................................................................
EnzChek® Protease Assay Kit *red fluorescence* *100-1000 assays* ................................................................................................................
epidermal growth factor (EGF) *from mouse submaxillary glands* .....................................................................................................................
epidermal growth factor, biotin-XX conjugate (biotin EGF) ..................................................................................................................................
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 488 streptavidin (Alexa Fluor® 488 EGF complex) .........................................
epidermal growth factor, biotinylated, complexed to Texas Red® streptavidin (Texas Red® EGF complex) .........................................................
epidermal growth factor, fluorescein conjugate (fluorescein EGF) .......................................................................................................................
epidermal growth factor, Oregon Green® 514 conjugate (Oregon Green® 514 EGF) ............................................................................................
epidermal growth factor, tetramethylrhodamine conjugate (rhodamine EGF) .......................................................................................................
Escherichia coli BioParticles® opsonizing reagent ................................................................................................................................................
Escherichia coli (K-12 strain) BioParticles®, Alexa Fluor® 488 conjugate .............................................................................................................
Escherichia coli (K-12 strain) BioParticles®, Alexa Fluor® 594 conjugate .............................................................................................................
Escherichia coli (K-12 strain) BioParticles®, BODIPY® FL conjugate ....................................................................................................................
Escherichia coli (K-12 strain) BioParticles®, fluorescein conjugate ......................................................................................................................
Escherichia coli (K-12 strain) BioParticles®, tetramethylrhodamine conjugate .....................................................................................................
Escherichia coli (K-12 strain) BioParticles®, Texas Red® conjugate .....................................................................................................................
Fc OxyBURST® Green assay reagent *25 assays* *3 mg/mL* ............................................................................................................................
fibrinogen from human plasma, Alexa Fluor® 488 conjugate ...............................................................................................................................
fibrinogen from human plasma, Alexa Fluor® 546 conjugate ...............................................................................................................................
fibrinogen from human plasma, Alexa Fluor® 594 conjugate ...............................................................................................................................
fibrinogen from human plasma, Oregon Green® 488 conjugate ...........................................................................................................................
FluoSpheres® collagen I-labeled microspheres, 1.0 µm, yellow-green fluorescent (505/515) *0.5% solids* ......................................................
FluoSpheres® collagen I-labeled microspheres, 2.0 µm, yellow-green fluorescent (505/515) *0.5% solids* ......................................................
formyl-Nle-Leu-Phe-Nle-Tyr-Lys, fluorescein derivative .......................................................................................................................................
gelatin from pig skin, Oregon Green® 488 conjugate ..........................................................................................................................................
gelatin from pig skin, fluorescein conjugate .........................................................................................................................................................
histone H1 from calf thymus, Alexa Fluor® 488 conjugate ...................................................................................................................................
hyaluronic acid, BODIPY® FL conjugate (BODIPY® FL hyaluronic acid) ...............................................................................................................
8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS; pyranine) .............................................................................................................
insulin, human, recombinant from E. coli, fluorescein conjugate (FITC insulin) *monolabeled* *zinc free* ........................................................
lactoferrin from human milk, Oregon Green® 514 conjugate ................................................................................................................................
lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 488 conjugate ............................................................................................................
N-levulinoyl-D-mannosamine (ManLev) ................................................................................................................................................................
N-levulinoyl-D-mannosamine, tetraacetate (ManLev tetraacetate) *5 mM solution in DMSO* .............................................................................
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 488 conjugate ....................................................................................
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 568 conjugate ....................................................................................
lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 594 conjugate ....................................................................................
lipopolysaccharides from Escherichia coli serotype 055:B5, BODIPY® FL conjugate ...........................................................................................
lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 488 conjugate .....................................................................................................
lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 568 conjugate .....................................................................................................
lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 594 conjugate .....................................................................................................
lipopolysaccharides from Salmonella minnesota, BODIPY® FL conjugate ............................................................................................................
low-density lipoprotein from human plasma (LDL) *2.5 mg/mL* ........................................................................................................................
low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 488 conjugate (Alexa Fluor® 488 AcLDL) *1 mg/mL* ......................................
low-density lipoprotein from human plasma, acetylated, BODIPY® FL conjugate (BODIPY® FL AcLDL) *1 mg/mL* ...........................................
692
Unit Size
Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels
10 µg
50 µg
100 mg
10 mg
25 mg
500 µg
500 µg
500 µg
500 µg
500 µg
1 mg
1 mg
5 mg
5 mg
1g
1g
5 x 1 mg
5 x 1 mg
5 x 1 mg
250 µL
1 kit
1 kit
100 µg
20 µg
100 µg
100 µg
20 µg
20 µg
20 µg
1U
2 mg
2 mg
10 mg
10 mg
10 mg
10 mg
500 µL
5 mg
5 mg
5 mg
5 mg
0.4 mL
0.4 mL
1 mg
5 mg
5 mg
1 mg
500 µg
1g
100 µg
1 mg
1 mg
25 mg
500 µL
100 µg
100 µg
100 µg
100 µg
100 µg
100 µg
100 µg
100 µg
200 µL
200 µL
200 µL
www.probes.com
Cat #
Product Name
L-3484
L-3483
L-3482
L-453
M-13440
M-23361
N-13195
N-23106
O-13291
P-3900
S-2860
S-23371
S-23372
S-2854
S-2851
S-2859
S-359
T-13342
T-23364
T-23365
T-13343
T-23362
T-23366
T-23363
T-2873
T-2871
T-13341
T-2872
T-2875
T-3163
T-1111
T-3166
T-13320
low-density lipoprotein from human plasma, acetylated, DiI complex (DiI AcLDL) *1 mg/mL* ...........................................................................
200 µL
low-density lipoprotein from human plasma, BODIPY® FL complex (BODIPY® FL LDL) *1 mg/mL* ..................................................................
200 µL
low-density lipoprotein from human plasma, DiI complex (DiI LDL) *1 mg/mL* .................................................................................................
200 µL
lucifer yellow CH, lithium salt ...............................................................................................................................................................................
25 mg
[Nle4, D-Phe7]-α-melanocyte-stimulating hormone, fluorescein conjugate ([Nle4, D-Phe7]-α-MSH, fluorescein conjugate) .........................................
25 µg
mucin from bovine submaxillary gland, Oregon Green® 488 conjugate ...............................................................................................................
5 mg
2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) ........................................................................................................
5 mg
6-(N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl)amino) -6-deoxyglucose (6-NBDG) ......................................................................................................
5 mg
OxyBURST® Green H2HFF BSA *special packaging* ............................................................................................................................................ 5 x 1 mg
N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium p-toluenesulfonate (TMAP-DPH) ............................................................
5 mg
Staphylococcus aureus BioParticles® opsonizing reagent ....................................................................................................................................
1U
Staphylococcus aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 488 conjugate ...................................................................
2 mg
Staphylococcus aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 594 conjugate ...................................................................
2 mg
Staphylococcus aureus (Wood strain without protein A) BioParticles®, BODIPY® FL conjugate ..........................................................................
10 mg
Staphylococcus aureus (Wood strain without protein A) BioParticles®, fluorescein conjugate ............................................................................
10 mg
Staphylococcus aureus (Wood strain without protein A) BioParticles®, unlabeled ...............................................................................................
100 mg
sulforhodamine 101 .............................................................................................................................................................................................
25 mg
transferrin from human serum, Alexa Fluor® 488 conjugate ................................................................................................................................
5 mg
transferrin from human serum, Alexa Fluor® 546 conjugate ................................................................................................................................
5 mg
transferrin from human serum, Alexa Fluor® 568 conjugate ................................................................................................................................
5 mg
transferrin from human serum, Alexa Fluor® 594 conjugate ................................................................................................................................
5 mg
transferrin from human serum, Alexa Fluor® 633 conjugate ................................................................................................................................
5 mg
transferrin from human serum, Alexa Fluor® 647 conjugate ................................................................................................................................
5 mg
transferrin from human serum, biotin-XX conjugate ............................................................................................................................................
5 mg
5 mg
transferrin from human serum, BODIPY® FL conjugate .......................................................................................................................................
transferrin from human serum, fluorescein conjugate ..........................................................................................................................................
5 mg
transferrin from human serum, Oregon Green® 488 conjugate ............................................................................................................................
5 mg
transferrin from human serum, tetramethylrhodamine conjugate ........................................................................................................................
5 mg
transferrin from human serum, Texas Red® conjugate ........................................................................................................................................
5 mg
N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43) .............................................................................
1 mg
N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414) .........................................................
5 mg
N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 4-64) ....................................................
1 mg
N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide
(FM® 4-64) *special packaging* .......................................................................................................................................................................... 10 x 100 µg
N-(3-triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide (FM® 2-10) .............................................................................
5 mg
1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) ..............................................................................
25 mg
N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 5-95) .................................................
1 mg
trypsin inhibitor from soybean, Alexa Fluor® 488 conjugate .................................................................................................................................
1 mg
Vybrant® Phagocytosis Assay Kit *250 assays* ..................................................................................................................................................
1 kit
zymosan A BioParticles® opsonizing reagent .......................................................................................................................................................
1U
zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 488 conjugate ..................................................................................................................
2 mg
zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 594 conjugate ..................................................................................................................
2 mg
zymosan A (S. cerevisiae) BioParticles®, BODIPY® FL conjugate .........................................................................................................................
10 mg
zymosan A (S. cerevisiae) BioParticles®, fluorescein conjugate ...........................................................................................................................
10 mg
zymosan A (S. cerevisiae) BioParticles®, Texas Red® conjugate ..........................................................................................................................
10 mg
zymosan A (S. cerevisiae) BioParticles®, unlabeled ..............................................................................................................................................
100 mg
T-7508
T-204
T-23360
T-23011
V-6694
Z-2850
Z-23373
Z-23374
Z-2844
Z-2841
Z-2843
Z-2849
Unit Size
16.2 Probes for Neurotransmitter Receptors
Because receptor-mediated signal transduction underlies much
of what occurs in cellular biochemistry and physiology,1,2 fluorescent receptor ligands can provide a sensitive means of identifying
and localizing some of the most pivotal molecules in cell biology.
Molecular Probes offers fluorescently labeled and unlabeled
ligands for various cellular receptors, ion channels and ion carriers. Many of these site-selective fluorescent probes may be used
on live or fixed cells, as well as in cell-free extracts. In particular,
we would like to highlight those ligands conjugated to the greenfluorescent Alexa Fluor 488, BODIPY FL and Oregon Green 514
dyes and the red-fluorescent Alexa Fluor 594 and Texas Red dyes,
which provide extremely bright signals and superior photostability. The high sensitivity and selectivity of these fluorescent probes
make them especially good candidates for measuring low-abundance receptors. Various methods for further amplifying detection
of these receptors are discussed in Chapter 6 and Chapter 7.
This section is devoted to our probes for neurotransmitter
receptors. Additional fluorescently labeled receptor ligands (including low-density lipoproteins, epidermal growth factors, transferrin and fibrinogen conjugates and chemotactic peptides) are
described in Section 16.1, along with other probes for studying
receptor-mediated endocytosis, as well as membrane markers of
Section 16.2
693