The Ion Indicator Company
FLUORESCENT ION INDICATOR HANDBOOK
FEATURE PRODUCTS
Asante NaTRIUM Green
- visible sodium indicator
SQI-Pr - a true sodium ionophore
Asante Potassium Green - visible potassium indicator
Fluo-2 - brighter and cheaper direct Fluo-4 replacement
Asante Calcium Green - extremely bright and leakage resistant
Asante Calcium Red - long wavelength red spectrum response
Asante Calcium nearIR - first near IR calcium indicator
Specialized forms of all calcium indicators
Letter to Researchers
Since 1992, TEFLabs’ mission has been to support more effective research. In recent years, our focus
has been to provide the widest range of fluorescent ion indicators and their specialized versions.
We are very pleased to introduce five indicator families which dramatically extend your testing
capabilities for sodium, potassium, and calcium ions.
Asante NaTRIUM Green is a new fluorescent sodium indicator that allows researchers to study sodium
using the same equipment and methods developed over the last twenty years to study calcium with
Fluo dyes. Operating in the visible spectrum, it loads readily to give a bright and sensitive response
to sodium. SQI-Pr is a true sodium ionophore, with a selectivity of 100:1, to complement Asante
NaTRIUM Green.
Asante Potassium Green is the potassium analog of Asante NaTRIUM Green, with the corresponding
ease of loading, brightness, and sensitivity to potassium.
We offer both low and high affinity versions of these sodium and potassium dyes.
TEFLabs has taken several approaches to improving calcium indicators by addressing the key
performance factors of brightness, loading, retention, accuracy, and response wavelength.
Asante Calcium Green is very bright and has extremely low leakage from cells, so that the use of
probenecid is not necessary for most experiments. In addition to supporting prolonged testing with
its no-leak properties, its high sensitivity combined with large dynamic range should make it the most
popular calcium indicator for both high throughput testing and cell research.
Asante Calcium Red exhibits red emission with a maximum at 650 nm and can be used either
ratiometrically or non-ratiometrically, depending on the wavelength of excitation. As with many
red dyes, the brightness is relatively low, but the long wavelength response, independent from
cellular autofluorescence and GFP emission, makes this indicator desirable for many applications.
Asante Calcium Near IR, an analog of this new indicator, can be excited at 635 nm with emission
at 690 nm.
Fluo-2 Medium Affinity is a direct replacement for Fluo-4, but it is brighter, easier to load, and less
expensive.
We are always pushing the limits of our research to provide more innovative optical tools for the future
of ion indicators. Please do not hesitate to engage us to address other ion indicator problems.
Thank you for your support over the years.
Sincerely,
A. Minta, Ph.D
Founder and Chairman
Front cover images left to right: Asante NaTRIUM Green-1 in HEK cells (page 7) [J. Kao, Univ. of Maryland
Medical School]; Asante NaTRIUM GREEN-1 in astrocytes with Ouabain stimulation of neurons (page 8)
[JY Chatton, Univ. of Lausanne]; Asante Calcium Red in neurons (page 15) [J Marks, Univ. of Chicago].
2
Introduction
1.1 General Information on Fluorescent Ion Indicators
4-5
Fluorescent Sodium Indicators
2.1 Introduction
2.2 Asante NaTRIUM GreenTM (ANG)
2.3 SBFI
2.4 Experimental Methods
6-10
Chapter Three Fluorescent Potassium Indicators 3.1 Introduction
3.2 Asante Potassium GreenTM (APG)
3.3 PBFI
3.4 Experimental Methods
11-13
Chapter Four Chapter Five
New Fluorescent Calcium Indicators 4.1 Introduction
4.2 Asante Calcium RedTM (ACR) 4.3 Asante Calcium Near-IRTM (ACnIR)
4.4 Asante Calcium GreenTM (ACG)
4.5 Fluo-2
4.6 Experimental Methods
14-21
Traditional Calcium Indicators 5.1 Introduction
5.2 Fura-2 5.3 Indo-1
5.4 Quin-2
5.5 Rhod-2
5.6 Non-Fluorescent Calcium Indicators
22-24
Chapter Six Special Versions of Calcium Indicators
6.1 Low Affinity Versions 6.2 Leakage Resistant Versions 6.3 Near Membrane Versions
25-29
Chapter Seven Other Ion Indicators 7.1 Magnesium
7.2 pH 7.3 Chloride 7.4 Zinc
30-33
Chapter Eight Ionophores
8.1 Sodium Ionophores
8.2 Potassium Ionophore
8.3 Calcium Ionophores
8.4 Experimental Methods
34-35
Chapter Nine Chapter Ten
Standard Fluorophores
36
Miscellaneous Products
10.1 Viability and Cytotoxicity Assay Products
10.2 Protein Kinase C Indicators
37
Other References
Distributors
Ordering Information
Price List
38
39
40
41-43
Contents
Chapter One Chapter Two Chapter 1
Introduction to Fluorescent Ion Indicators
TEFLabs is a global leader in ion indicators owing to the unique expertise of its founder, Dr. Akwasi Minta. Dr. Minta was a
member of Dr. Roger Tsien’s small team of pioneers in the early 1980s that introduced the fluorescent ion indicators Fura -2,
Rhod-2, Indo-1, SBFI, PBFI, Fluo-2 and Fluo-3. Dr. Minta commercialized these indicators at Molecular Probes and at TEFLabs,
and expanded the repertoire by introducing indicators for zinc, chloride, and other analytes. In 2006, TEFLabs set a focus on
producing a new generation of enhanced ion indicators. The exceptional fruits of this effort are presented in this handbook.
TEFLabs is wholly dedicated to the continued creation of the best fluorescent ion indicators.
1.1 General Information on Fluorescent Ion Indicators
Indicator Form
We provide most of our indicators in two forms:
The acetoxymethyl (AM) ester form, first introduced in 1981 by Dr. Tsien1, 2 is non-invasive and is the most popular method for
loading fluorescent ion indicators into cells. The phenolic and carboxylic acid functions of the molecule are derivatised as AM
esters. These esters make the molecule hydrophobic enough to be membrane permeant. Once inside the cell, non-specific
esterases, found in almost all cell types, hydrolyze the esters back to the polyanionic form necessary for water-solubility;
retention in the cell; and, for sensing ions.
The water-soluble salt (e.g., K+ or TMA+) is the active form of the indicator. It is available for calibration purposes or for invasive
loading, such as microinjection into cells3 or loading through a whole-cell patch electrode.4
TEFLabs sells AM forms of dyes in aliquots of 1 mg, 500 µg, or 50 µg. For testing purposes we offer 2 x 50 µg units. We also
offer the AM forms in dry dimethylsulfoxide (DMSO) solution (sealed under argon) and provide PluronicTM F127, a surfactant
that aids in dispersing the dye in the aqueous loading buffer.
AM Ester Loading Guidelines
When cells are incubated with the AM ester of an indicator, the AM ester permeates into the cell and is hydrolyzed by
intracellular esterases to yield the active form of the indicator, which becomes trapped and accumulated in the cell. For loading,
a 1-10 mM stock solution of the AM ester is prepared using anhydrous DMSO and stored at -20ºC. In order to prevent the
deterioration of the AM esters that occurs with repeated thawing and freezing, we recommend that 1 mg or 500 µg quantities
be divided into aliquots containing 50 µg each. Loading is usually performed in a serum-free culture medium with the AM ester
at a final concentration ranging from 1-10 µM. Pluronic F-127 may be added to the loading medium to aid dispersal of the
AM esters; it is typically used at a concentration of < 0.1% (wt/vol). For ease of use, a stock solution of Pluronic F-127 should be
made in dry DMSO at a concentration of 20% (wt/vol). The required volumes of the DMSO stock solutions of the AM ester and
of the Pluronic should be premixed and then dispersed into aqueous medium for loading. The cells can be incubated at room
temperature or 37ºC (although quality of loading is often better at room temperature), and the time of incubation typically
ranges from 30 to 60 minutes. After loading, the cells should be washed at least once with fresh serum-free culture medium to
minimize extracellular background fluorescence.
Notes:
1) If the loading medium is buffered with bicarbonate, then loading should be done under a 5% CO2 atmosphere to
prevent alkalinization of the medium through loss of CO2;
2) if serum-containing medium is used for loading, then the loading concentration of AM ester may need to be increased
to compensate for binding of AM esters to serum proteins;
3) in some cases (particularly when the AM ester has a molecular weight near or exceeding 1000), the loaded cells
may require a further incubation in medium without AM ester for 20 – 60 minutes to allow complete processing of
the AM ester by intracellular esterases;
4) there are also procedures for performing “no wash” assays; and
5) incubation conditions can vary among cell types and among indicators and ideally should be optimized for each
cell type.
Protocol guidelines for our new dyes are provided at www.teflabs.com.
Fluorescence and ratiometry
Two types of spectral response are possible when an ion binds to a fluorescent indicator. First, the fluorescence intensity changes
with essentially no significant change in the shape of the fluorescence spectrum (Figure. 1.1). Second, the shape of the emission
(Figure 1.2) or excitation (Figure 1.3) spectrum changes so that the optimal wavelength of excitation or emission changes.
4
39000 nM!
1350 nM!
Fluorescence Intensity (a.u.)!
3000000!
2500000!
602 nM!
Asante !
Calcium Red!
351 nM!
225 nM!
150 nM!
2000000!
100 nM!
65 nM!
38 nM!
1500000!
17 nM!
0 nM!
1000000!
500000!
Excitation !
at 540 nm!
0!
550!
600!
650!
Wavelength (nm)!
700!
750!
Figure 1.1 Fluorescence emission traces of a titration of Asante Ca2+ Red (excitation at 540 nm)
showing a calcium-dependent enhancement of
fluorescence but not a shift of wavelength.
Figure 1.2 Fluorescence emission traces of Asante Calcium Figure 1.3
2+
Red (excitation at 488 nm) acquired at zero (Ca -free) and
saturating (Ca2+-bound) concentrations of Ca2+. The spectral
change can be used for emission ratiometry. [J. Kao, Univ of
Maryland Medical School]
Spectral changes as a function of ion concentration can be analyzed by means
of a Hill plot or by non-linear regression to obtain the dissociation constant, Kd,
of the indicator.5
When there is a ion-dependent change in the wavelength of maximal emission
or excitation, the ion’s concentration can be determined from a ratio of the
fluorescence intensities acquired at two distinct wavelengths.12 The advantage
of such ratiometric measurements is that the ratioing of intensities eliminates
potential artifacts due to variable degree of cell loading, loss of intracellular
indicator by leakage or photobleaching, changes of cell thickness during an
experiment, as well as certain changes in detector sensitivity.
The remarkable benefits of ratiometry make it an attractive goal in the
design of an indicator. One of our new products is a novel ratiometric dye,
Asante Calcium Red (ACR, Figure 1.2). ACR is the first true emission ratiometric
indicator that is excited at visible wavelengths (488 nm). By exciting at 540
nm, ACR can also be used non-ratiometrically (Figure 1.1).
For an indicator that is not ratiometric, a large fluorescence dynamic range
is vital, i.e., the difference between the minimal fluorescence (Fmin, at zero ion
concentration) and the maximal fluorescence (Fmax, at saturating ion concentration)
should be as large as possible. This ensures high detection sensitivity so that even
small changes in ion concentration translate into fluorescence changes that are
easily measurable. We are excited to introduce Asante Calcium Green (ACG),
with the largest dynamic range measured to date (Fmax/Fmin = 220). (Figure 1.4,
see Chapter 4 for more information about ACG).
Fluorescence excitation traces of
a titration of Fura-2 showing calcium-dependent
excitation ratiometry.
Figure 1.4 Fluorescence emission spectra of a calcium
titration of Asante Ca2+ Green, showing an exceptional
dynamic range (Fmax/Fmin = 220). [J. Kao, Univ. of Maryland
Medical School]
Kd and sensitivity
Calibration is necessary to obtain the dissociation constant (Kd) of each indicator dye. Physical conditions such as temperature,
pH, ionic strength, and solution viscosity, as well as interactions of the dye with cellular constituents such as proteins, all affect Kd.
In practice, the Kd in the cell is typically higher than the value obtained in vitro. Therefore, if precise calibration is required, then
the Kd should be determined in the cells under study.
The Kd’s of our ion indicators vary, so it is important to select the indicator based on the ion concentration range expected
from an experiment. In general, the Kd is the midpoint of the concentration range to which the indicator is sensitive. A
practical rule-of-thumb is that indicators are typically responsive to ion concentrations ranging from Kd/30 to 30×Kd (or, in
logarithmic terms, pKd ± 1. 5).
Chapter 1 - Introduction
5
Chapter 2
Fluorescent Sodium Indicators
2.1 Introduction
There is a large difference in the sodium ion (Na+) concentration inside and outside the cell (5-40 mM intracellular; 120-450 mM
extracellular, depending on organism). This concentration gradient is used to power nutrient uptake, to regulate concentrations
of other intracellular ions and solutes, and to generate and transmit electrical impulses in excitable cells such as nerve and
muscle. These functions are so important that organisms devote a major part of their metabolic energy to maintaining the
sodium gradient.7,8 The low intracellular Na+ concentration requires that a Na+ indicator have the sensitivity to measure any
small changes that occur. Moreover, intracellular potassium ion (K+) concentration is typically high (in excess of 100 mM);
therefore a Na+ indicator should respond selectively to Na+, not K+.
Table 2.1 lists the properties of sodium indicators offered by TEFLabs. The titration curves for Asante Natrium Green (Figures
2.4 and 2.5) should help in selecting a sodium indicator.
Table 2.1 Sodium Indicators
Catalog
Number
MW
Absorbance Excitation
(g/mol)
(nm)
(nm)
Emission
(nm)
Kda
(mM)
Solubility
ANG-1 (TMA+ salt)
3520
1100
517
488-517e
540
92c
H2O
ANG-1(AM)
3500+
1100
469
N/Ad
N/Ad
N/Ad
DMSO
ANG-2 (TMA+ salt)
3522
1100
517
488-517e
540
20c
H2O
ANG-2 (AM)
3502
1100
469
N/Ad
N/Ad
N/Ad
DMSO
SBFI (K+ salt)
0032
991
339
505
4b
H2O
SBFI (AM)
0030+
1127
379
N/Ad
N/Ad
DMSO
340/380
(high/low Na+)
N/Ad
a
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. These Kd’s were measured in simple aqueous buffers as a
guideline to the scientist, who should then calibrate the dye the cells under study.
b
The excitation maximum is 517 nm; 488 nm excitation gives 40% of the maximum.
c
The titrations to determine Kd were performed by mixing known amounts of 1 M TMACl, 10 mM MOPS, pH 7.1 and 1M NaCl, 10 mM MOPS, pH 7.1, each solution with equal concentrations
of the indicator. No corrections were made for increasing ionic strength or for quenching by chloride.
d
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Na+ sensitive salt form..
e
The titration to determine Kd for SBFI was performed in 100 mM TMACl, 10 mM MOPS with the addition of sodium chloride. No corrections were made for increasing ionic strength or
quenching by chloride.
Two sets of dyes are represented in Table 2.1: SBFI and the two forms of ANG.
SBFI, introduced in 1989,9 has been the dye most frequently used for measuring Na+ until now. Unfortunately, SBFI requires
excitation by UV light and is difficult to load. Previous efforts to replace SBFI by visible wavelength indicators have yielded
Sodium GreenTM, CoroNaTM Red, and CoroNaTM Green, but they have problems associated with ineffectiveness in the cell,
inability to detect small Na+ changes, and leakage from cells. (Sodium GreenTM, and CoroNaTM are trademarks of Molecular
Probes division of Life Technologies.)
Our new Na+ indicators, ANG-1 (Asante NaTRIUM GreenTM 1) and ANG-2 ( Asante NaTRIUM GreenTM 2), display a range of
desirable characteristics, including high sensitivity, high loading efficiency, and excellent brightness and dynamic range. These
indicators are excited by visible light and thus permit sodium studies to be conducted on equipment already in use for calcium
high throughput screening and for cell and ion channel studies.
Table 2.2 compares SBFI’s performance with Asante NaTRIUM Green.
6
Table 2.2
SBFI
ANG1/ANG2
Kd
4 mM
92 mM/ 20 mM
Excitation
340 and 380 nm
488 to 517 nm
Emission
500 nm
540 nm
Brightness
Dim
Very Bright
<3
29
AM ester loading
Difficult
Easy
Photobleaching
Sensitive
Resistant
Dynamic Range (Fmax/Fmin)
2.2 Asante NaTRIUM GreenTM
ANG-1
Asante NaTRIUM Green 1 is a visible wavelength fluorescent indicator with a useful dynamic range for measuring cytosolic Na+
concentrations. Unlike SBFI, it loads readily into cells and is excited by visible light. Although ANG-1 excites maximally at 517
nm, its exceptional brightness enables excitation at the standard 488 nm settings used for the Fluo calcium indicators. Moreover,
it works well for 2-photon excitation with near-infrared light. ANG-1 is also remarkable in its resistance to photobleaching and
leakage. It is available as the tetramethylammonium (TMA+) salt and the acetoxymethyl (AM) ester. The TMA+ salt form is
provided for patch-clamping and other direct methods of introducing the dye into cells, as well as for in vitro calibration of the
dye. Figures 2.1-2.3 demonstrate the indicator’s performance in the cell:
(I) Screening mode (Figure 2.1): HEK293 cells expressing TRPV1 channels were loaded with ANG-1. Capsaicin, an agonist
for TRPV1 channels, which conduct Na+ and Ca2+, was used to stimulate Na+ influx. The capsaicin-evoked rise in ANG-1
fluorescence is robust. [J. Kao, Univ. of Maryland Medical School]
(II) Flow cytometry and wide-field microscopy mode (Figure 2.2): REF52 cells were loaded with ANG-1, and the Na+ ionophore
gramicidin was applied to promote Na+ influx. The resulting rise in intracellular Na+ concentration caused a corresponding increase in
ANG-1 fluorescence. [J. Kao, Univ. of Maryland Medical School]
Figure. 2.1 ANG-1 in HEK293 cells
Chapter 2 - Fluorescent Sodium Indicators
Figure. 2.2 ANG-1 in REF52 cells
7
Chapter 2
Figure 2.3 - ANG-1 in astrocytes
(III) Confocal microscope mode (Figure 2.3): Astrocytes were
loaded with ANG-1 and then ouabain, a sodium pump inhibitor,
was used to block Na+ extrusion from the cell. The second frame
shows the consequent increase in ANG-1 fluorescence from
baseline (first frame) after only three minutes of treatment with
ouabain. [JY Chatton, Univ. of Lausanne]
Baseline
Ouabain (3 min.)
The following is an ANG-1 (AM) cell loading procedure for the experiments of Figures 2.1 and 2.2. (This procedure should
not be treated as a general loading protocol for ANG-1. Please refer to Chapter 1 for general loading guidelines.)
(i) 1-5 μM of ANG-1 (AM) is an average concentration.
(ii) The sample is prepared with ~75 ppm Pluronic F-127 for dispersing the dye into the incubator buffer.
(iii) For these experiments, the buffer consisted of HCO3- DMEM with 10% fetal bovine serum (FBS) and cells were kept
under 5% CO2 atmosphere.
(iv) Loading time varied from 45 minutes to 70 minutes.
(v) The experiments were conducted in HBSS.
[J. Kao of Univ. of Maryland Medical School] See section 2.4 for experimental details.
ANG-2
Asante NaTRIUM GreenTM 2 (ANG-2) is a higher affinity analog of ANG-1, as demonstrated by comparing the fluorimetric
emission sodium chloride titrations of ANG-1 (Figure 2.4) and ANG-2 (Figure 2.5), with identical excitation and emission wavelengths. It retains the desirable properties of easy loading, slow leakage, and good photostability.
4000000!
Fluorescence Intensity (a.u.)!
3000000!
Asante NaTRIUM Green 2!
(ANG-2)!
56.2 mM!
31.6 mM!
17.8 mM!
2000000!
9.99 mM!
5.62 mM!
1500000!
3.16 mM!
1.78 mM!
1000000!
Excitation at 517 nm!
Figure 2.4
316 mM
1500000
100 mM
56.2 mM
31.6 mM
17.8 mM
1000000
9.99 mM
5.62 mM
3.16 mM
500000
1.78 mM
0 mM
0 mM!
0
550!
575!
600!
Wavelength (nm)!
625!
650!
525
Excitation at 517 nm
Figure 2.5
Two example applications of ANG-2 in cells are shown in Figures 2.6 and 2.7.
8
178 mM
Asante NaTRIUM Green 1
(ANG-1)
1.00 mM!
500000!
525!
562 mM
100 mM!
2500000!
0!
2000000
181 mM!
Fluorescence Intensity (a.u.)
3500000!
315 mM!
550
575
600
Wavelength (nm)
625
650
Figure 2.6 Response of intracellular ANG-2 to
Na+ influx facilitated by SQI-Pr: REF52 fibroblasts
loaded with ANG-2 (through incubation with AM ester)
were maintained in Hanks’ Balanced Salt Solution
(HBSS). SQI-Pr is a Na+ ionophore that promotes Na+/
H+ exchange across the cell membrane, and thus causes
Na+ influx. Upon application of 40 μM SQI-Pr, the
fluorescence of intracellularly-loaded ANG-2 increased
steadily, reflecting a rise in intracellular [Na+]. Further
addition of 20 μM amphotericin B, a polyene natural
product that increases membrane permeability to all
the common monovalent ions (Na+, K+, H+ and Cl-), gave
a small additional increment of indicator fluorescence,
as expected. This experiment demonstrates the utility
of ANG-2 as a sodium indicator, and the efficacy of
SQI-Pr as a sodium ionophore. The response shown is
the average from 30 cells. [J. Kao, Univ. of Maryland Medical
School]
Figure 2.7 Response of intracellular ANG-2 to Na+
influx: REF52 fibroblasts were loaded with ANG-2
(through incubation with AM ester) and maintained in
145 mM N-methyl-D-glucamine (NMG) gluconate. To
deplete cells of Na+ and K+, the cells were treated with
50 μM amphotericin-B (a polyene microbial metabolite
that markedly increases membrane permeability to
monovalent ions; e.g., Na+ K+, H+ and Cl-). Increments of
NaCl were added to raise sodium concentration ([Na+])
in the extracellular medium to various levels (5 , 15, 45,
and 145 mM, as indicated in the figure). After each
increment, as the intracellular and extracellular [Na+]
equilibrated, a corresponding increase in intracellular
indicator fluorescence was observed. At the end of the
experiment, an aliquot of KCl was added to raise [K+] to
145 mM (typical cytosolic value). The added K+ actually
decreased the fluorescence of ANG-2 modestly. The
response shown is the average from 25 cells. [J. Kao, Univ.
Figure 2.6
Figure 2.7
of Maryland Medical School]
The following is an ANG-2 (AM) cell loading procedure for the experiments of Figures 2.6 and 2.7. (This
procedure should not be treated as a general loading protocol for ANG-2. Please refer to Chapter 1 for
general loading guidelines.)
(i) 2 μM of ANG-2 (AM) is an average concentration.
(ii) The sample is prepared with ~75 ppm Pluronic for dispersing the dye into the incubator buffer.
(iii) The buffer consisted of HCO3- DMEM with 10% fetal bovine serum (FBS) and kept under 5% CO2 atmosphere.
(iv) Loading time varied from 45 minutes to 70 minutes.
(v) The experiments were conducted in HBSS.
[J. Kao of Univ. of Maryland Medical School] See section 2.4 for experimental details.
Chapter 2 - Fluorescent Sodium Indicators
9
2.3 SBFI
SBFI (Figure 2.8) is available in the cell impermeant K+ salt form and the cell permeant
acetoxymethyl (AM) ester form. It is an ultraviolet (340 and 380 nm) excitation
ratiometric Na+ indicator. It suffers from inefficient loading through the AM form
and from relatively low brightness. Although SBFI has been the best available Na+
indicator up to now, most scientists will want to switch to Asante Natrium Green for
greatly improved performance.
Figure 2.8 SBFI
The loading guidelines for SBFI are shown in Chapter 1, page 4.
2.4 Experimental Methods
Figure 2.1 Methods: HEK293 cells permanently overexpressing TRPV1, loaded 70-80 minutes at room temperature
with 5 μM ANG-1 AM + 75 ppm Pluronic F-127 in HCO3- -buffered DMEM containing 10% fetal bovine serum (FBS),
under 5% CO2 atmosphere. Experiments were conducted in HBSS. Excitation wavelength 488 nm. [J. Kao, Univ. of Maryland
Medical School]
Figure 2.2 Methods: REF52 cells loaded 1-2 hours with 5 μM ANG-1 AM + 75 ppm Pluronic F-127 in HCO3- -buffered
DMEM containing 10% fetal bovine serum (FBS), under 5% CO2 atmosphere. Experiments were conducted in HBSS.
Excitation wavelength 488 nm. Gramicidin D, a Na+ ionophore was used to transport Na+ from the medium into the cell.
[J. Kao, Univ. of Maryland Medical School]
Figure 2.6 Methods: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM Asante Natrium
Green-2 AM ester in bicarbonate-buffered Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum, under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075% (w/v) of the nonionic
surfactant, Pluoronic F-127. The cells were then transferred into HBSS. SQI-Pr was prepared as a 20 mM stock solution
in DMSO; amphotericin B was prepared as a 50 mM solution in DMSO. Appropriate volumes of the stock solutions
were added to the HBSS bathing the cells to achieve the desired final concentrations used in the experiment. Indicator
fluorescence was excited at 488 nm; fluorescence images were acquired with a cooled CCD camera. [J. Kao, Univ. of
Maryland Medical School]
Figure 2.7 Methods: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 µM Asante Natrium
Green-2 AM ester in bicarbonate-buffered Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal
bovine serum, under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075% (w/v) of the nonionic
surfactant, Pluoronic F-127. The cells were then transferred into 145 mM NMG gluconate (pH 7.4). Amphotericin B was
prepared as a 50 mM solution in DMSO; 1000-fold dilution into aqueous medium gave the desired final concentration
used in the experiment. Appropriate volumes of 4.0 M NaCl or KCl were added to achieved the desired concentrations in
the aqueous medium. Indicator fluorescence was excited at 488 nm; fluorescence images were acquired with a cooled CCD
camera. [J. Kao, Univ. of Maryland Medical School]
10
Chapter 3
Fluorescent Potassium Indicators
3.1 Introduction
The importance of the potassium ion (K+) is coupled to the sodium ion (Na+), because the cell expends a major part of its
metabolic energy maintaining the concentrations of Na+ and K+ within the cell. Intracellular concentration ranges are 10-40 mM
for Na+ and 120-400 mM for K+. Extracellular concentration ranges are 4-40 mM for K+ and 120-400 mM for Na+.
In the absence of a K+ indicator, efforts have been directed to using indirect techniques to measure or detect K+, including10
• analogs like thallium or cesium to monitor K+ fluxes;
• the pH indicator BCECF AM and the ionophore nigericin in flow cytometry studies;
• combinations of ion selective carriers;
• ion-channel mediated K+ fluxes with membrane potential changes registered by voltage sensitive dyes; and
• fiber-optic sensors for K+ with pH sensitive dyes.
These alternate techniques were necessary because the previously reported fluorescent potassium indicator PBFI9 requires UV
excitation and suffers from poor loading and poor brightness. The new TEFLabs indicators, Asante Potassium Green 1 and 2
(APG-1 and APG-2) successfully address these problems. The properties of the K+ indicators are shown in Table 3.1.
Table 3.1 Potassium Indicators
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
(mM)a
APG-1 (TMA+ salt)
3620
1100
517
488 to 517b
540
54c
H2O
APG-1 (AM)
3600
1100
469
N/Ad
N/Ad
N/Ad
DMSO
APG-2 (TMA+ salt)
3622
1100
517
488 to 517b
540
18c
H2O
APG-2 (AM)
3602
1100
469
N/Ad
N/Ad
DMSO
PBFI (TMA+ salt)
0022
1175
336
N/Ad
340/390
500
5e
H2O
PBFI (AM)
0020
1171
369
N/Ad
N/Ad
DMSO
(high/low Na+)
N/Ad
Kd
Solubility
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. These Kd’s were measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the dye the cells under study.
b
The excitation maximum is 517 nm; 488 nm excitation gives 40% of the maximum.
c
The titrations to determine Kd were performed in 140 mM TMACl, 10 mM MOPS, pH 7.1 with the addition of potassium chloride. No corrections were made for increasing
ionic strength or for quenching by chloride.
d
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its K+ sensitive salt form.e The titration
to determine Kd for PBFI was performed in 100 mM TMACl, 10 mM MOPS with the addition of potassium chloride. No corrections were made for increasing ionic strength
or for quenching by chloride.
a
Table 3.2 compares PBFI and Asante Potassium Green.
Table 3.2
PBFI
APG1/APG2
Kd
5 mM
54 mM/ 18 mM
Excitation
340 and 390 nm
488 to 517 nm
Emission
500 nm
540 nm
Brightness
Dim
Very Bright
Dynamic range (Fmax/Fmin)
<3
12
AM ester loading
Difficult
Easy
Photobleaching
Sensitive
Very Stable
11
3.2 Asante Potassium GreenTM
APG-1
Asante Potassium Green 1 (APG-1) is a fluorescent indicator with a useful Kd for measuring cytosolic K+ concentration. It loads
readily and is excited by visible light. Although non-ratiometric, its large fluorescence dynamic range allows sensing of even
small changes in K+ concentration. Optimal excitation occurs at 517 nm, but the indicator can also be excited at the conventional
wavelength of 488 nm. APG-1 works well with 2-photon excitation at near-infrared wavelengths and is quite resistant to
photobleaching. Like its sodium counterpart, it is useful for confocal microscopy, flow cytometry, and screening. A test showing
the response of APG-1 to K+ is shown in Figure 3.1.
Figure 3.1 Response of intracellular APG-1 to K+: REF52 fibroblasts
loaded with APG-1 (through incubation with AM ester) were maintained
in 145 mM N-methyl-D-glucamine (NMG) gluconate. To deplete cells
of K+ and Na+, the cells were treated with 50 µM amphotericin-B (a
polyene microbial metabolite that markedly increases membrane
permeability to monovalent ions; e.g., Na+ K+, H+ and Cl-). Increments
of KCl were added to raise potassium concentration ([K+]) in the
extracellular medium to various levels (5 , 15, 45, and 145 mM, as
indicated in the figure). After each increment, as the intracellular and
extracellular [K+] equilibrated, a corresponding increase in intracellular
indicator fluorescence was observed. At the end of the experiment, an
aliquot of NaCl was added to raise [Na+] to 10 mM, a typical cytosolic
value. The added Na+ only slightly affected the fluorescence of APG1. The response shown is the average from 45 cells. [J. Kao, Univ. of Figure 3.1
Maryland Medical School]
The following is an APG-1 (AM) cell loading procedure for the experiment of Figure 3.1. (This procedure should not be
treated as a general loading protocol for APG-1. Please refer to Chapter 1 for general loading guidelines.)
(i) 1-5 μM of APG-1 (AM) is an average concentration.
(ii) The sample is prepared with ~75 ppm Pluronic F-127 for dispersing the dye into the incubator buffer.
(iii) The buffer consisted of HCO3- DMEM with 10% fetal bovine serum (FBS) and kept under 5%
CO2 atmosphere.
(iv) Loading time varied from 45 minutes to 70 minutes.
(v) The experiments were conducted in HBSS.
[J. Kao of Univ. of Maryland Medical School] See section 3.4 for experimental details.
APG-2
APG-2 is a higher affinity analog of APG-1, as demonstrated by comparing the fluorimetricemission potassium chloride
titrations of APG-1 (Figure 3.2) and APG-2 (Figure 3.3), with identical excitation and emission wavelengths. It retains the
desirable properties of easy loading, slow leakage, and good photostability.
4000000!
567 mM!
180 mM!
100 mM!
2500000!
56.2 mM!
2000000!
17.8 mM!
31.6 mM!
9.99 mM!
1500000!
5.62 mM!
1000000!
1.78 mM!
3.16 mM!
1.00 mM!
500000!
0!
525!
Excitation at 517 nm!
Figure 3.2
12
0 mM!
550!
575!
600!
Wavelength (nm)!
5000000!
625!
650!
Fluorescence Intensity (a.u.)!
Fluorescence Intensity (a.u.)!
3000000!
177 mM!
5500000!
321 mM!
Asante!
Potassium Green 1 !
(APG-1)!
315 mM!
6000000!
1000 mM!
3500000!
100 mM!
Asante!
Potassium Green 2!
(APG-2)!
4500000!
4000000!
56.2 mM!
31.6 mM!
3500000!
17.8 mM!
3000000!
9.99 mM!
2500000!
5.62 mM!
2000000!
3.16 mM!
1500000!
1.78 mM!
1000000!
1.00 mM!
0 mM!
500000!
0!
525!
Excitation at 517 nm!
550!
575!
600!
Wavelength (nm)!
625!
650!
Figure 3.3
Chapter 3 - Fluorescent Potassium Indicators
Chapter 3
3.3 PBFI
PBFI (Figure 3.4) is the K+ analog of the Na+ indicator SBFI. Unfortunately,
the K+/Na+ selectivity of PBFI is less than the Na+/K+ selectivity of
SBFI. Despite its relatively poor K+/Na+ selectivity, PBFI has been used
with some success in cellular experiments because the intracellular K+
concentration is much greater than that of Na+.
PBFI shares SBFI’s shortcomings of ultraviolet excitation, difficulty in
loading the AM ester, and poor brightness. Asante Potassium Green
successfully addresses all of these problems.
Figure 3.4 PBFI
PBFI is available in the cell impermeant K salt form and the cell permeant acetoxymethyl (AM) ester form. While PBFI has been used for
a number of years, we encourage researchers to use Asante Potassium
Green indicators for improved performance.
+
The loading guidelines are shown in Chapter 1.
3.4 Experimental Methods
Figure 3.1 Methods: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 μM Asante Potassium Green
1 AM ester in bicarbonate-buffered Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum,
under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075% (w/v) of the nonionic surfactant, Pluoronic
F-127. The cells were then transferred into 145 mM NMG gluconate (pH 7.4). Amphotericin B was prepared as a 50 mM
solution in DMSO; 1,000-fold dilution into the aqueous medium gave the desired final concentration of 50 μM. Appropriate
volumes of 4.0 M KCl or NaCl were added to achieve the desired concentrations in the aqueous medium. Indicator fluorescence
was excited at 488 nm; fluorescence images were acquired with a cooled CCD camera.
[J. Kao, Univ. of Maryland Medical School]
13
Chapter 4
New Fluorescent Calcium Indicators
4.1 Introduction
The study of the function of calcium ions (Ca2+) inside cells is one of the most dynamic areas of modern cell biology. In common
parlance, a transient rise in the cytosolic free Ca2+ concentration is referred to as a “Ca2+ signal”. Ca2+ signaling is essential in
diverse biological processes, including mitosis, vesicular secretion (e.g., release of various neurotransmitter and hormones), motility
(e.g., muscle contraction), and learning and memory (e.g., long-term potentiation and depression of synaptic transmission).
Techniques for the measurement and manipulation of Ca2+ are therefore crucial and have advanced rapidly, largely as a result
of the invention of fluorescent calcium indicators.
The commonly used fluorescent Ca2+ indicators today (Fluo, Fura, and Indo) were invented in the 1980s, with
little improvement over the last 25 years. Scientists at TEFLabs have now developed new Ca2+ indicators that aim
to remedy shortcomings in the old generation of indicators.
TEFLabs is committed to improving Ca2+ indicators and supporting scientists by
• providing visible-wavelength indicators that offer greatly enhanced brightness and extended dynamic range Asante Calcium Green (ACG): Fmax/Fmim = 220, brighter than Fluo-4 by more than 3-fold, and leakage-resistant;
• providing improved long-wavelength and near-infrared indicator families - Asante Calcium Red (ACR) and
Asante Calcium NearIR (ACnIR);
• providing all Ca2+ indicator families in versions with enhanced performance characteristics, including low
affinity, leakage resistance, and near-membrane Ca2+ sensing versions; and
• continuing innovation in fluorescent Ca2+ indicators.
Table 4.1 summarizes TEFLabs’ new and traditional fluorescent Ca2+ indicator families. Within each family, TEFLabs offers
indicators for a range of affinities and enhanced-performance versions. The availability of multiple versions within each family
permits the researcher to select an indicator that is most appropriate for a particular experiment.
Table 4.1 - TEFLabs Calcium Indicator Families
New Calcium Indicators
Indo-1
Rhod-2
Quin2
Fluo-2
Fura-2
ACG
346
UV
excitation
550
(nm)
333/353
491
UV
340/380
488
to 517
UV
405/475
emission
578
(nm)
495
515
510
540
emission
ratiometric
no
excitation
no
excitation
no
bright
brightness
bright
verydim
bright
bright
exceptional
Indo-1
ACR
346or 540
488
UV
405/475
650
emission
emission
(optional)
bright
reasonable
Traditional Calcium Indicators (Chapter 5)
Rhod-2
ACnIR
Fluo-2
ACG Quin2
ACRFura-2
550 635
333/353488 or340/380
491 excitation
488 to 517
540
(nm)
UV
UV
578 690
515
no no
emission
540
(nm)
495
650 510
no ratiometric no excitation emission
excitation
(optional)
bright
exceptionaldim
reasonable very bright
brightness
reasonable
bright
ACnIR
Indo-1
Rhod-2
635346
UV
550
491
690
405/475
578
515
no
emission
no
no
reasonable
bright
bright
Ca2+ indicators that are excited at visible wavelengths have advantages over UV-excited indicators. Whereas UV light can
cause cell damage, visible light is generally biologically benign. Moreover, much less cellular autofluorescence is excited by
visible light than by UV light. The ready availability of low-cost lasers that emit visible light makes the visible indicators ideal
for confocal microscopy and flow cytometry.
14
Fluo
b
very br
4.2 Asante Calcium Red (ACR)
Asante Calcium Red emits fluorescence at long wavelengths, where there is virtually no interference from cellular autofluorescence.
Exciting at 488 nm produces dual fluorescence emission at 650 and 525 nm, enabling emission ratiometry. ACR can also be
excited at 540 nm to generate fluorescence emission at 650 nm, which can be used for non-ratiometric measurement. Table 4.2
summarizes the properties of ACR.
Table 4.2 - ACR Properties
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
ACR (K salt)
3020+
1000
555
488b or
540
525/650b or
650
400
H2O
ACR (AM)
3000+
1200
472
N/Ac
N/Ac
N/Ac
DMSO
+
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Exciting at 488 nm produces dual emission ratiometry; increasing [Ca2+] produces strongly increasing emission intensity at 650 nm and slightly decreasing emission
intensity at 525 nm.
c
Once the AM ester form permeates the cell membrane, non-specific esterases hydrolyze the AM esters to yield the indicator in its Ca2+-sensitive salt form.
a
39000 nM!
1350 nM!
3000000!
Fluorescence Intensity (a.u.)!
2500000!
602 nM!
351 nM!
Asante !
Calcium Red!
225 nM!
150 nM!
100 nM!
2000000!
65 nM!
38 nM!
17 nM!
1500000!
0 nM!
1000000!
500000!
0!
550!
Excitation at 540 nm!
Figure 4.1
600!
650!
Wavelength (nm)!
700!
750!
ACR in Non-ratiometric mode
When excited at 540 nm (Figure 4.1), ACR emits maximally at 650 nm; it shows
a 50-fold increase in intensity on going from the Ca2+-free to the Ca2+-bound
form. The red fluorescence of ACR is not as intense as the green fluorescence
of Fluo dyes, but it is useful for many applications.
Figure 4.2 Fluorescence image of ACR in cultured embryonic rat hippocampal neurons. The cells were loaded with 3 μM ACR (AM) for 30 minutes in
culture medium and then incubated in dye-free medium for 30 minutes before
imaging. [J Marks, Univ. of Chicago.]
Figure 4.3 Relative change in ACR fluorescence in the cell bodies of selected
neurons in response to stimulation with NMDA (300 µM) in glycine-containing,
Mg2+-free saline. [J Marks, Univ. of Chicago.]
Figure 4.3
Figure 4.2
Chapter 4 - New Fluorescent Calcium Indicators
15
Chapter 4
ACR in ratiometric mode
Ratiometry offers ease of calibration and enables quantitative measurement by eliminating variables such as indicator
concentration, photobleaching, indicator leakage, and cell thickness. Furthermore, a red-emitting indicator significantly reduces
interference from cellular autofluorescence, which typically occurs at shorter wavelengths (e.g., flavin nucleotide cofactors, FMN
and FAD, emit green fluorescence peaking at ~540 nm).
Until now, only two useful ratiometric Ca2+ indicators have been known: Fura-2 and Indo-1. Fura -2 exhibits excitation ratiometry,
while Indo-1 is principally used for emission ratiometry. Both of these indicators require cell-damaging UV excitation and
expensive UV-transmitting optics.
Table 4.3 - Comparison of ratiometric calcium dyes
Fura-2
Indo-1
ACR
Kd
200 nM
250 nM
400 nM
Excitation
340/380
350
488
Emission
505
485-410
650/525
Brightness
very bright
bright
less bright
Dynamic range
3x
4x
25x
AM ester loading
normal
normal
normal
Photostability
stable
poor
more stable
The following is an ACR (AM) cell loading procedure for the experiments of Figures 4.4 and 4.5. (This
procedure should not be treated as a general loading protocol for ACR. Please refer to Chapter 1 for
general loading guidelines.)
(1) Prepare stock solution of 1 – 10 mM ACR (AM) in DMSO
(2) Incubate cells in 3 – 10 µM ACR (AM) in aqueous medium containing ~0.02% Pluronic F-127 (premix the requisite
volume of ACR (AM) stock in DMSO with the desired volume of 15% wt/vol stock of Pluronic in DMSO before
dispersal into aqueous medium)
(3) Incubate 45 – 60 minutes at room temperature
(4) Wash cells and maintain cells in fresh medium for ~40 minutes at room temperature to ensure complete intracellular
hydrolysis of the AM ester
[J Kao, Univ. of Maryland Medical School] See section 4.6 for experimental details.
16
Table 4.3 compares Asante Calcium Red’s performance to that of traditional ratiometric Ca2+ indicators.
When excited at 488 nm, Asante Calcium Red can be used for dual emission ratiometry (Figure. 4.4). Upon Ca2+ binding, ACR
shows a marked increased in 650 nm fluorescence (25 fold), with a concomitant slight decrease in 525 nm fluorescence. The extent
of decrease at 525 nm can vary from cell to cell, but simply having a second emission independent of the strongly Ca2+-sensitive
emission enables ratiometry. For example, in rat cardiac myocytes (Figure 4.5) the 525 nm fluorescence intensity remains essentially
constant; this enables the Ca2+-sensitive intensity at 650 nm to be ratioed against a constant 525 nm intensity.
Figure 4.4 Emission ratiometric imaging with ACR of an ATP-evoked calcium transient in a rat vagal sensory neuron.
Line traces show the time-course plots of fluorescence intensities at 650 and 525 nm, as well as the intensity ratio. Images on
top corespond to the time points marked on the line traces. [J Kao, Univ. of Maryland Medical School]
Figure 4.5 Emission ratiometric imaging of rat cardiac myocyte loaded with ACR and stimulated electrically. Line traces
show the time-course plots of fluorescence intensities at 650 and 525 nm, as well as the intensity ratio. Images on the left correspond to time points marked on the lines traces. [B Hagen, J Lederer, J Kao, Univ. of Maryland Medical School]
Figure 4.4
Figure 4.5
4.3 Asante Calcium NearIR (ACnIR)
Until now, there have been no Ca2+ indicators that emit in the near-IR wavelength
range. TEFLabs has successfully created a near-IR Ca2+ indicator, Asante Calcium
NearIRTM (ACnIR). ACnIR is a nonratiometric indicator with excitation maximum at
630 nm and fluorescence emission peaking at 690 nm. ACnIR is available in the
cell impermeant K+ salt form and the cell permeant acetoxymethyl (AM) ester form.
A fluorescence emission titration (Figure 4.6) shows indicator response to Ca2+.
Cellular experiments will be posted on our website (www.teflabs.com) as they
become available. Table 4.4 summarizes ACnIR properties.
Figure 4.6
Chapter 4 - New Fluorescent Calcium Indicators
17
Chapter 4
Table 4.4 - ACnIR Properties
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
ACnIR (K+ salt)
3320+
1100
635
630
690
350
H2O
ACnIR (AM)
3300+
1300
313b
N/Ac
N/Ac
N/Ac
DMSO
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
ACnIR (AM) is colorless.
c
Once the AM ester form permeates the cell membrane, non-specific esterases hydrolyze the AM esters to yield the indicator in its Ca2+-sensitive salt form.
a
4.4 Asante Calcium Green (ACG)
In the past, the nonratiometric Fluo dyes have been very useful and popular because they have a large dynamic range (Fmax/
Fmin ≈ 100). Asante Calcium Green (ACG) is much brighter than the Fluo dyes with an even larger dynamic range: Fmax/Fmin =
220 (Figure 4.7), where the relevant quantum efficiencies are: Ca2+-free Q = 0.00225, Ca2+-bound Q = 0.495. Figure 4.8
compares the quantum efficiency of Ca2+-bound ACG to fluorescein.
ACG’s high fluorescence quantum efficiency makes it the brightest Ca2+ indicator with the highest fluorescent dynamic range
to date. This exceptional brightness and dynamic range allows ACG to report both small and large Ca2+ signals with good
signal-to-noise.
Figure 4.9: ACG responses to neuronal Ca2+ transients evoked by depolarizations ranging in duration from 1 to 1000
msec. ACG was introduced into rat vagal sensory (nodose ganglion) neurons through a whole-cell patch electrode (electrode
filling solution containing 50 µM ACG K+salt). The holding potential of the neuron was Vm = −70 mV. The neuron received a
series of step depolarizations from −70 to +10 mV that ranged in duration from 1 − 1000 msec. Intracellular Ca2+ signals
evoked by the depolarizing steps were recorded as increases in indicator fluorescence. For ease of quantitative comparison,
fluorescence signals are reported as fractional change of fluorescence relative to baseline fluorescence intensity (ΔF/F0). [J Kao,
Univ. of Maryland Medical School]
Figure 4.7
Figure 4.8
Figure 4.9
TEFLabs plans to offer multiple versions of ACG with a range of Kds. Improved methods of non-invasive cell loading are being
developed. Please check our website (www.TEFLabs.com) for loading protocols.
Table 4.5 summarizes ACG properties.
18
Table 4.5 - ACG Properties
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
ACG (K+ salt)
3704+
1200
518
517
540
135b
H2O
ACG (AM)
3700+
1400
470
N/Ac
N/Ac
N/Ac
DMSO
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Kd as determined in aqueous buffers; intracellular Kd may double. The combination of a very large dynamic range and high affinity gives a high initial brightness that
can still increase greatly upon saturating with calcium.
c
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
4.5 Fluo-2
Fluo-2 is one of the original Fluo series of Ca2+ indicators invented by Drs.
Roger Tsien and Akwasi Minta in the 1980s.11 Whereas Fluo-3 and Fluo-4 were
commercialized, Fluo-2 was not (Figure 4.10). Subsequently, Fluo-2 Medium Affinity
(Fluo-2 MedAff) has been found to be much brighter (1.5x) than Fluo-4 in cellular
experiments, as shown in Figures 4.11 and 4.12. The increased brightness of Fluo2 MedAff is partly attributable to its superior loading in most cell types.
Table 4.6 shows a comparison of Fluo-2 MedAff and Fluo-4 properties. The
pKa of Fluo-2 MedAff is slightly higher than Fluo-4. Although a lower pKa was
assumed necessary when the Fluo dyes were invented, experimental data has
established that Fluo-2’s pKa is not a limitation. Please see our website (www.
teflabs.com) for more information on the history of the Fluo dyes and their pKa’s.
MedAff
For improved performance over Fluo indicators, TEFLabs recommends Asante
Calcium Green (substantially enhanced brightness and dynamic range) and, for
Figure 4.10 - Fluo-2 MedAff, 3, 4
some applications, Asante Calcium Red (fluorescence emission at much longer
wavelength).
MedAff
Figure 4.11 - Fluo-2 MedAff, Fluo-3, and Fluo-4 brightness
Chapter 4 - New Fluorescent Calcium Indicators
Figure 4.12 - Fluo-2 Med Aff and Fluo-4 cell loading
19
Chapter 4
Table 4.6 - Fluo-2 MedAff versus Fluo-4 comparison
Fluo-4
Fluo-2 MedAff
Kd (nM)
350
400
pKa
5.6
6.2
Excitation (nm)
488
488
Emission (nm)
516
515
Brightness
1x
1.5x
Dynamic range
100x
150x
AM ester loading
Normal
Easier
Photostability
Stable
Stable
The general properties of Fluo-2 are shown in Table 4.7.
Table 4.7 Fluo-2 properties
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
Fluo-2 MedAff (K+ salt)
0204+
877
490
490
515
390
H2O
Fluo-2 MedAff (AM)
0200+
1047
452
N/Ab
N/Ab
N/Ab
DMSO
Fluo-2 HighAff (K+ Salt)
0224
891
490
490
515
230
H2O
Fluo-2 HighAff (AM)
0220+
1061
455
N/Ab
N/Ab
N/Ab
DMSO
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
The protocol for using Fluo-2 is the same that is used for Fluo-4.
Typical loading protocol for Fluo-2
(1) Prepare stock solution of 1 – 10 mM Fluo-2 (AM) in DMSO
(2) Dilute AM stock solution to 1 – 5 μM in aqueous medium and incubate cells for 30 – 60 minutes at room temperature.
(If necessary, premix Pluronic F-127 stock solution (15% wt/vol in DMSO) with the desired volume of Fluo-2 (AM) stock
solution before dispersal into aqueous medium.)
(3) Wash/transfer cells into fresh medium for experimentation (for “wash-free” assays, washing may be omitted).
20
4.6 Experimental Methods
Figure 4.2 Methods: Cells were perfused in a flow- through chamber and imaged on an inverted Nikon microscope with
a 40x, 1.35 NA objective. Filtered Light from a xenon lamp was used to illuminate the cells; the excitation filter had a center
wavelength of 572 nm (35 nm wide bandpass). Fluorescence was passed through an emission filter centered at 632 nm (60
nm wide bandpass) before it was imaged with a cooled CCD camera. Scale bar indicates 20 μm. [J Marks, Univ. of Chicago.]
Figure 4.3 Methods: Images were obtained every 20 seconds. Regions of interest were drawn around the somata, and
mean intensities within the regions recorded as a function of time. Percent change of fluorescence at each time was calculated
with respect to an average of the first five images.
Figure 4.4 Methods: ACR in rat vagal sensory neurons. The inferior vagal (nodose) ganglia from a Sprague-Dawley rat
were dissociated enzymatically. The yield of nodose neurons was suspended in Leibovitz L-15 medium supplemented with
10% (v/v) fetal bovine serum and penicillin-streptomycin and plated onto No. 1 glass coverslips. Neurons were incubated
for 50 minutes at room temperature with 3 μM ACR (AM) ester dispersed with 0.015% (wt/vol) Pluoronic F-127 in L-15
medium. Thereafter, the cells were washed and maintained in fresh L-15 medium for 40 minutes at room temperature to permit
intracellular enzymatic hydrolysis of the AM ester to proceed to completion. [J Kao, Univ. of Maryland Medical School]
Figure 4.5 Methods: ACR in rat cardiac myocytes. Acutely dissociated rat cardiac myocytes were incubated for 45 minutes
at room temperature in Tyrode’s solution containing 10 μM ACR (AM) and 0.02% (wt/vol) Pluronic F-127. The cells were then
transferred to fresh Tyrode’s solution and allowed to stand for an additional 45 minutes to ensure complete hydrolysis of the
AM ester. [B Hagen, J Lederer, J Kao, Univ. of Maryland Medical School]
Figure 4.9 Methods: Nodose ganglia were dissected from adult Sprague-Dawley rats and enzymatically dissociated to
yield nodose ganglion neurons (NGNs). Neurons were voltage-clamped in the whole-cell configuration; 50 μM ACG K+ salt
was added to the intracellular solution in the patch electrode. Normal Locke’s solution was used as the extracellular medium.
Confocal fluorescence images were acquired on a laser-scanning confocal microscope at 2.56 frames per second. The indicator
was excited at 488 nm. [J Kao, Univ. of Maryland Medical School]
Chapter 4 - New Fluorescent Calcium Indicators
21
Chapter 5
Traditional Calcium Indicators
5.1 Introduction
The first group of useful fluorescent calcium indicators12 Fura-2, Indo-1, and Quin-2 are excitable only with ultraviolet light. They
have been very useful because of their ratiometric properties, despite the damaging energy from the UV light and intrinsic
autofluorescence from the cell. All of these indicators display a shift in fluorescence upon binding calcium, and a ratio of the two
wavelengths can be used to effect calibration. TEFLabs offers Fura-2, Indo-1, and Quin-2 in the form of their cell impermeant
K+ salt form and the cell permeant acetoxymethyl (AM) ester form. Table 5.1 summarizes the properties of these UV-excitable
calcium indicators.
Table 5.1 - UV-excitable Calcium Indicators
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
Fura-2 (AM)
0102
1002
371
N/Ab
N/Ab
N/Ab
DMSO
Fura-2 (K+ SALT)
0104
832
354
505
145
H2O
Indo-1 (AM)
0105
1010
356
N/Ab
N/Ab
N/Ab
DMSO
Indo-1 (K+ SALT)
0107
840
346
346
230
H2O
Quin-2 (AM)
0114
830
354
N/Ab
N/Ab
N/Ab
DMSO
Quin-2 (K+ SALT)
0115
694
353
495
60
H2O
340/380
(high/low Ca2+)
333/353
(high/low Ca2+)
405/475
(high/low Ca2+)
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
5.2 Fura-2
Fura-2 (Figure 5.1) is one of the first commercial fluorescent calcium indicators introduced by Dr. Tsien and produced by Molecular Probes in 1986. It is now so common in the field that it appears in standard molecular cell biology textbooks. Fura-2
exhibits excitation ratiometry at 340 nm/380 nm with emission at 505 nm. There
are thousands of Fura-2 references in the scientific literature.
Figure 5.1
5.3 Indo-1
Indo-1 (Figure 5.2) was introduced at the same time as Fura-2 in 1986. It is also
ratiometric, but different in mode. Rather than being excitation ratiometric, it exhibits an emission ratio at 475 nm/ 405 nm when excited at 346 nm. Unlike Fura-2, it
has a tendency to photobleach. It has also found a great amount of applications,
exemplified by the large number of literature publications.
22
Figure 5.2
5.4 Quin-2
Quin-2 (Figure 5.3) was the first fluorescent calcium indicator made by Dr. Tsien after he
invented BAPTA as the precursor to all calcium indicators. It is still used today, but the higher
quantum yield of Fura-2 has all but replaced it as an excitation ratio indicator. It has an
excitation ratio of 383 nm/ 353 nm with emission at 495 nm.
Figure 5.3
TEFLabs sells Quin-2 in the cell-impermeant K+ salt and the cell-permeant AM esters
forms.
The properties of these dyes including their Kds can be found in Table 5.1
5.5 Rhod-2
TEFLabs sells the longer wavelength Rhod-2 in the cell-impermeant K+ salt and the cellpermeant AM esters forms. It has a tendency to sequester in mitochondria. Table 5.2 lists
its properties.
Figure 5.4
Table 5.2
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
Rhod-2 (K+ salt)
0121
869
547
550
578
570
H2O
Rhod-2 (AM)
0119
1124
551
N/Ab
N/Ab
N/Ab
DMSO
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
5.6 Non-Fluorescent Calcium Indicators
As summarized in Figure 5.5, TEFLabs’ non-fluorescent calcium indicators are BAPTA13 and its bromine and fluorine derivatives.
BAPTA [1,2,-bis(o-aminophenoxy)ethane-N,N,-N’,N’, tetra-acetic acid] forms the basis of all the fluorescent calcium ion indicators.
It was derived from EGTA [ethylene glycol bis(β-aminoethyl ether) N,N,N’,N’ tetra-acetic acid] to create a selectivity of 105 for
Ca2+ over Mg2+.
BAPTA (X=Y=H) itself is now used essentially for buffering calcium, but the lower affinity fluoro
derivatives – difluoro (X=F, Y=H) and tetrafluoro11 (BAPTA FF, X=Y=F), have been a good source
for studying high concentration of calcium using 19F NMR. 5,5’-DimethylBAPTA or “MAPTA”
(X=CH3, Y=H) is the highest affinity BAPTA of all. DibromoBAPTA (X=Br, Y=H) has an
intermediate affinity and has been used extensively to study calcium mobilization, spatial
buffering, and calcium shuttling in many cells29-31. All the BAPTA derivatives are available
Figure 5.5
in the cell impermeant potassium salts or free acids and as cell permeant acetoxymethyl
esters. The properties are shown in Table 5.3.
Chapter 5 - Other Calcium Indicators
23
Chapter 5
Table 5.3 Non-Fluorescent Calcium Indicators
Catalog Number
MW (g/mol)
Kda (nM)
Solubility
BAPTA (K+ salt)
0101
629
160
H2O
BAPTA (AM)
0100
765
N/Ab
DMSO
BAPTA FF (K+ salt)
0148
701
65000 (65 µM)
H2O
BAPTA FF (AM)
0147
837
N/Ab
DMSO
Dibromo BAPTA (K+ salt)
0162
787
16000 (1.6 µM)
H2O
Dibromo BAPTA (AM)
0161
922
N/Ab
DMSO
MAPTA (K+ salt)
0164
657
40
H2O
MAPTA (AM)
0163
793
N/Ab
DMSO
Difluoro BAPTA (K+ salt)
0123
665
635
H2O
Difluoro BAPTA (AM)
0122
801
N/Ab
DMSO
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
TEFLabs offers K+ salt and AM versions of Half-BAPTA, N-(o-methoxyphenyl)iminodiacetic acid. Half-BAPTA is a good
control for potential effects of BAPTA and BAPTA-based calcium indicators that are not related to Ca2+ binding. [Kao et al.,
2010. Meth. Cell Biol. 99:113-152] Structurally, Half-BAPTA is essentially half of BAPTA. It has extremely low affinity for
Ca2+ (Kd » 3 mM) and is expected to mimic BAPTA and BAPTA-based indicators in most chemical respects except for the
ability buffer Ca2+ intracellularly.
The AM ester of Half-BAPTA provides an easy way to load Half-BAPTA into cells. It can also be used as a control for AM
ester loading in general.
24
Chapter 6
Special Versions of Calcium Indicators
TEFLabs calcium indicators are available in special forms that allow scientists to tailor the dyes to different experiments. These
versions include Low Affinity (LowAFF) for measuring Ca2+ concentrations [Ca2+]; Leakage Resistant (LeakRes), to improve
indicator in the cell; and Near Membrane, (NearMem), to secure the indicator at the cell membrane.
6.1 Low Affinity Versions
Low Affinity (LoAff, formerly designated as FF) Ca2+ indicators enable asurement of high [Ca2+]. They have a potential advantages over their high affinity counterparts. They cause reduced buffering of intracellular Ca2+ and consequently, reduced
perturbation of Ca2+ signals. Thus, they allow more temporally accurate measurements of faster, short-lived Ca2+ signals. In the
past, researchers haveused APTRA-based magnesium analogs for such applications, but they have encountered interference
from the binding of magnesium ions. TEFLabs provides low affinity versions of BAPTA-based calcium ion indicators to minimize
this interference.
Figure 6.1 Fura-2 LowAff
Figure 6.2 Indo-1 LowAff
The first low affinity indicators from TEFLabs were Fura-2FF (now Fura-2
LowAff, Figure 6.1), and Indo-1FF (now Indo-1 LowAff, Figure 6.2). We
have expanded our offering to include Low Affinity versions for Asante
Calcium Red, Fluo-2, and Rhod-2. Please check our website (www.teflabs.com) for the availability of Asante Calcium Green LowAff. The
general properties of these indicators can be found in Table 6.1 below.
Table 6.1
Catalog MW Absorbance
Number (g/mol)
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
N/Ab
N/Ab
N/Ab
DMSO
Fura-2 LowAff (AM)
0135
1038
370
Fura-2 LowAff (K+ salt)
0137
853
360
Indo-1 LowAff (AM)
0138
1046
348
N/Ab
Indo-1 LowAff (K+ salt)
0140
862
348
346
Rhod-2 LowAff (AM)
0159
1146
551
Rhod-2 LowAff (K+ salt)
0160
891
3070
340/380
2+
(high / low Ca )
25000 (25 μM)
505
at 37° C
H2O
N/Ab
DMSO
(high / low Ca )
26000 (26 μM)
at 34° C
H2O
N/Ab
N/Ab
N/Ab
DMSO
550
550
577
19000 (19 μM)
H2O
1000
555
488 or 540c
525/650c or 650
to be
determined
H2O
3050+
1200
472
N/Ab
N/Ab
N/Ab
DMSO
3720
1100
518
488 to 517d
540
to be
determined
H2O
3716+
1400
469
N/Ab
N/Ab
N/Ab
DMSO
Fluo-2 LowAff (K+ salt)
0244
913
491
490
515
to be
determined
H2O
Fluo-2 LowAff (AM)
0240+
1083
455
N/Ab
N/Ab
N/Ab
DMSO
Asante Calcium Red
LowAff (K+ salt)
Asante Calcium Red
LowAff (AM)
Asante Calcium Green
LowAff (K salt)
Asante Calcium Green
LowAff (AM)
+
N/Ab
400/485
2+
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
25
TEFLabs offers the even lower affinity nitro versions of Fura-2 and Indo-1 on a custom
synthesis basis.
For those who would still prefer to use the APTRA-based indicators (Furaptra or Fura-2
Mg, Indo-1 Mg, etcetera), please refer
to section 7.1.
6.3a Fura-2
Figure 6.3b Furaptra
The low affinity Ca2+ indicator selectivity
over Mg2+ is illustrated in Figure 6.3.18
Fura-2 (Figure 6.3a) is saturated at levels
as low as 2 µM Ca2+. This saturation may
be avoided by using the higher Kd Furaptra
or Fura-2 LowAff (Fura-2 FF indicators).
However, Furaptra (Figure 6.3b) cannot
distinguish Mg2+ from Ca2+, and so it reports
a high mitochondrial (M) [Ca2+]. Fura-2
LowAff (Figure 6.3c) reports the correctly
low mitochondrial concentration for Ca2+
because Mg2+ minimally contributes to its
fluorescence response.
Figure 6.4
Figure 6.4 shows fluorescence excitation
traces (emission at 510 nm) of a Ca2+
titration of Fura-2 LowAff.
Figure 6.3c Fura-2 LowAf
6.2 Leakage Resistant Versions
Leakage resistance (LeakRes) versions have a special appendage that increases
the intracellular retention – well in excess of an hour.
The first leakage resistant Ca2+ indicators, Fura-2 LeakRes (Fura-PE3, formerly
Figure 6.5) and Indo-1 LeakRes (Indo-PE3, formerly Figure 6.6) were developed
by Minta and Poenie in the early 1990s.19-21 Compared to the corresponding nonLeakRes versions, the LeakRes indicators are identical in fluorescence properties
but are retained in the cell for hours. In addition to Fura and Indo, TEFLabs has
applied this unique technology to our new indicators Asante Calcium Red and
Fluo-2. (Asante Calcium Green has a very high leakage resistance in its base
form.) The LeakRes indicators are cell impermeant.
TEFLabs plans to offer dextran versions of our calcium indicators in the near
future. At this time, in lieu of dextran, we offer leakage resistant versions for
injection in cell-impermeant salt forms. In particular, we offer our ultra-resistant
Asante Calcium Green.
Figure 6.5 Fura-2 LeakRes
Figure 6.6 Indo-1 LeakRes
Figure 6.7 Retention of Fura-2 LeakRes and leakage of Fura-2: 322 T
lymphoma cells wer loaded with either Fura-2 or Fura-2 LeakRes (formerly FuraPE3) and set in calcium buffer. Leakage of Fura-2 or Fura-2 LeakRes (denoted
as PE3) into the exterior calcium buffer resulted in increased fluorescence overall.
This fluorescence was plotted over time.21
Figure 6.7
26
Chapter 6 - Special Versions of Calcium Indicators
Chapter 6
Figure 6.8
Figure 6.8 Intracellular fluorescence demonstrating retention of Fura--2 LeakRes and leakage of Fura-2: The top
row shows images of BPV cells loaded with Fura-2 LeakRes, whereas the bottom row shows BPV cells loaded with Fura2. Images were taken at every 20 minutes. Fura-2 cells show loss of significant fluorescence by T = 40 minutes. Fura-2
LeakRes cells retain fluorescence even at T = 100 minutes.
The leakage-resistant Ca2+ indicators has have been used frequently since their introduction in 1994; several literature
references are provided as a guide to their use.5,19,22-29 Table 6.2 shows the properties of TEFLabs’ LeakRes indicators.
Table 6.2 - Leakage Resistant Indicators
Emission
(nm)
Kda
(nM)
Solubility
505
145
(224 at 37°C)
H2O
N/Ab
N/Ab
N/Ab
DMSO
346
N/Ab
N/Ab
N/Ab
DMSO
1062
346
346
260
H2O
3150+
1400
472
N/Ab
N/Ab
N/Ab
DMSO
3170
1200
555
488 or 540c
525/650c or 650
to be
determined
H2O
3704
1100
518
488 to 517d
540
135e
H2O
Asante Calcium Green (AM)
3700+
1300
469
N/Ab
N/Ab
N/Ab
DMSO
Fluo-2 LeakRes (AM)
0230+
1317
455
N/Ab
N/Ab
N/Ab
DMSO
0234
1113
490
490
515
to be
determined
H2O
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Fura-2 LeakRes (K+ salt)
0110
832
354
Fura-2 LeakRes (AM)
0108
1258
371b
Indo-1 LeakRes (AM)
0144+
1266
0146
Indo-1 LeakRes
+
(K salt)
Asante Calcium Red LeakRes
(AM)
Asante Calcium Red LeakRes
(K+ salt)
Asante Calcium Green
(K+ salt)
Fluo-2 LeakRes
(K+ salt)
Excitation
(nm)
340/380
2+
(high/low Ca )
408/475
(high/low Ca2+)
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
27
6.3 Near Membrane Versions
The near membrane (NearMem) Ca2+ indicators have a special lipophilic anchor that tethers the dye to the lipid membrane,
so that it can report on changes in [Ca2+] ocurring near the membrane. A caveat on using NearMem Ca2+ indicators is that,
being anchored in the cell membrane means that they can sense Ca2+ influx near its source - namely the Ca2+ channels in the
cell membrane. [Ca2+] near these sites of Ca2+ influx can rise to high micromolar levels, which can saturate the response of the
indicators. Design of low affinity near membrane dyes is ongoing.
The first near-membrane fluorescent calcium indicators were based on Fura-2 and
Indo-1 and were developed by Minta and Poenie.20-21 Unlike other hydrophobic near
membrane indicators, TEFLabs NearMem indicators have a hydrophilic spacer that
allows the lipid anchor to lodge in the membrane while the Ca2+ indicator is poised
above the membrane surface to sense Ca2+. Figures 6.9 and 6.10 show the structures
of Fura-2 NearMem (formerly FFP-18)21, and Indo-1 NearMem (formerly FIP-18)21.
Figure 6.9 Fura-2 NearMem
The same near membrane design is applied to our new indicators, Asante Calcium Red
and Fluo-2. We offer the NearMem indicators as their cell impermeant potassium
salts and in the cell permeant AM ester form.
The application of Fura-2 NearMem in neutrophils is an example of near-membrane
study32. Figure 6.11A shows the distribution of Fura-2 NearMem in the plasma
membrane30. Figure 6.11B is a plot of fluorescence intensity of the indicator as a
function of the distance from the cell membrane. Figure 6.12 shows a fluorescence
excitation (emission set at 500 nm) titration of Fura-2 NearMem with Ca2+.
The AM ester loading guideline in Chapter 1 may need to be adjusted for higher
concentrations of these NearMem indicators and longer loading times, depending
on the cell type. The properties can be seen in Table 6.3.
Figure 6.10 Indo-1 NearMem
Figure 6.12
Figure 6.11
28
Chapter 6 - Special Versions of Calcium Indicators
Chapter 6
Table 6.3 - Near Membrane Indicators
Catalog
Number
MW
(g/mol)
Absorbance
(nm)
Excitation
(nm)
Emission
(nm)
Kda
(nM)
Solubility
Fura-2 NM (AM)
0125
1296
364
N/Ab
N/Ab
N/Ab
DMSO
Indo-1 NM (AM)
0127
1303
346
N/Ab
N/Ab
N/Ab
DMSO
Indo-1 NM (K+ salt)
0129
1134
346
346
450
H2O
Asante Calcium Red NearMem
3220
1300
555
488 or 540c
525/650c or
650
to be
determined
H2O
Asante Calcium Red NearMem
(AM)
3200+
1500
472
N/Ab
N/Ab
N/Ab
DMSO
Fluo-2 NearMem (K+salt)
0254
1185
490
490
515
to be
determined
H2O
Fluo-2 NearMem (AM)
0250+
1355
455
N/Ab
N/Ab
N/Ab
DMSO
408/475
(high/low Ca2+)
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator the cells under study.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Ca2+ sensitive salt form.
a
6.4 Experimental Methods
Figure 6.8 Methods: BPV cells, adhered to coverslips, were loaded with Fura-2 LeakRes(AM) or Fura-2(AM) as described in
Materials and Methods. Cells were mounted in a Sykes-Moore chamber and placed on a water-jacketed holder of a Zeiss IM35 microscope. The temperature was maintained at 37οC in the sample chamber by a thermostatically controlled circulating
water bath. Images were acquired with a Hammamatsu SIT camera and a Photon Technology Image Master illumination and
acquisition system. Images of the same microscope field were recorded at 360 nm excitation at 20-min intervals beginning
immediately after cells were washed. Camera gain and intensifier voltages were set based on the brightness of cells at the
first time point and maintained constant thereafter. Between the acquisition of light was blocked by a shutter. (A-F) The upper
series of photographs shows the pattern of fluorescence change for Fura-2 LeakRes loaded BPV cells. The lower series of
photographs (G-L) shows the corresponding changes in Fura-2 loaded BPV cells.
29
Chapter 7
Other Ion Indicators
7.1 Magnesium
Magnesium ions are essential in mediating processes such as enzymatic reaction, muscular contraction,
hormonal secretion, and DNA synthesis. The divalent magnesium cation, although in the same group
as calcium, is a smaller atom. It is therefore not surprising that truncated versions of calcium indicators
work well for magnesium. BAPTA-based indicators have a selectivity of about 105, for Ca2+ over
Mg2+. Trimming the binding site by roughly half greatly reduces affinity for Ca2+, but still allows
binding of the much smaller Mg2+. Thus, APTRA33 (Figure 7.1), approximately half BAPTA, is a nonfluorescent Mg2+ indicator, and the structural basis for all our other magnesium indicators. In view of Figure 7.1
this design, we offer Mg2+ versions of almost all our Ca2+ indicator versions.
These Mg2+ indicators have also gained popularity as very low affinity indicators. The practical
problem with their use is one of distinguishing the contribution of Mg2+ in Ca2+ measurements. We
therefore offer special low-affinity Ca2+ indicators that are not based on APTRA and that are highly
selective for binding Ca2+ over Mg2+ (See section 6.1.)
APTRA
Long wavelength magnesium indicators
TEFLabs offers the long-wavelength FLUO-2 Mg (Figure 7.2), Asante Magnesium RedTM, and Asante
Magnesium GreenTM. Fluo-2 Mg is cell impermeant. The loading guidelines are described in Chapter 1 Figure 7.2 Fluo-2 Mg
and the properties are shown in Table 7.1.
Table 7.1 - Long Wavelength Magnesium Indicators
Catalog
Number
MW
ABSmax
(nm)
EXmax
(nm)
EMmax
(nm)
Kda
(mM)
Solubility
Fluo-2 Mg (AM)
0047
851
460
N/Ab
N/Ab
N/Ab
DMSO
Fluo-2 Mg (K+ salt)
0048
715
505
505
525
5.1
H2O
Asante Calcium Red
Mg (AM)
0048
715
505
505
525
5.1
H2O
0048
715
505
505
525
5.1
H2O
0048
715
505
505
525
5.1
H2O
0048
715
505
505
525
5.1
H2O
Asante Calcium Red
Mg (K+ salt)
Asante Calcium
Green Mg (AM)
Asante Calcium
Green Mg (K+ salt)
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator to the experimental sytem being used.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM esters to yield the indicator in its Mg2+-sensitive salt form.
a
30
UV Magnesium Indicators
TEFLabs offers the UV-excitable Mg2+ indicators that correspond to Fura-2 (Fura-2 Mg, Figure 7.3) and Indo-1 (Indo-1 Mg,
Figure. 7.4). Fura-2 Mg was first invented by Raju et al34 as “Furaptra”.
The properties of Fura-2 Mg and Indo-1 Mg are shown in Table 7.2 below.
Figure 7.4 Indo-1 Mg
Figure 7.3 Fura-2 Mg
Table 7.2 - UV Magnesium Indicators
Catalog
Number
MW
(g/mol)
ABSmax
(nm)
EXmax
(nm)
EMmax
(nm)
Kda
(mM)
Solubility
Fura-2 Mg (AM)
0040
723
366
N/Ab
N/Ab
N/Ab
DMSO
Fura-2 Mg (K+ salt)
0042
587
360
490
1.9
H2O
Indo-1 Mg (AM)
0043
731
354
N/Ab
N/Ab
N/Ab
DMSO
Indo-1 Mg (K+ salt)
0046
595
349
343
2.7
H2O
360/369
(high/low Mg2+)
417/480
(high/low Mg2+)
The dissociation constant (Kd) is sensitive to pH, temperature, viscosity, ionic strength, competing ions, and cellular interactions. This Kd was measured in simple aqueous
buffers as a guideline to the scientist, who should then calibrate the indicator to the experimental sytem being used.
b
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM esters to yield the indicator in its Mg2+-sensitive salt form.
a
7.2 pH
Knowledge of cytosolic pH (pHi) is essential in many cellular studies. The most popular
indicator for studying pHi is BCECF42 (Figure 7.5). Its pKa of 6.98 is ideal because all
cells have pHi near 7.0, with physiological changes of no more than a few tenths of
a pH unit. BCECF has an excitation spectrum with a maximum at 507 nm and an isoexcitation point at 438 nm (at which the fluorescence emission is independent of pH).
Intracellular pH can therefore be estimated by measuring the ratio of the fluorescence
intensities at 500 and 450 nm. We offer BCECF in the form of cell-impermeant free
Figure 7.5 BCECF
acid and the cell-permeant acetoxymethyl esters. (See Table 7.3)
Chapter 7 - Other Indicators
31
Chapter 7
Table 7.3 - pH Indicators
Catalog
Number
MW
(g/mol)
ABSmax
(nm)
EXmax
(nm)
EMmax
(nm)
pKaa
Solubility
BCECF (AM)
0061+
821
507
N/Ab
N/Ab
N/Ab
DMSO
BCECF
(Free acid)
0060
520
507
500/450
(hi/lo pH)
531
7
H2O
The acid dissociation constant (pKa) is sensitive to temperature, viscosity, ionic strength, and cellular interactions. This pKa was measured in simple aqueous buffers as a
guideline to the scientist, who should then calibrate the indicator to the experimental sytem being used.
b
Once the acetate and AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the acetates and AM esters to yield the indicator in
its pH sensitive (salt) form.
c
The free acid should be dissolved at pH > 7.
a
7.3 Chloride
Chloride (Cl-) ion fluxes play an important role in cellular regulatory, absorptive, and secretory processes. The known Cl- indicators
work by collisional quenching of fluorescence36, an entirely different mechanism than that for the cation indicators described
earlier. Quenching is not accompanied by spectral shift, so ratio measurements are not possible. Current Cl- indicators are
based on the 6-methoxyquinoline fluorophore. TEFLabs offers two Cl- indicators, SPQ 37,38 (Figure 7.6)and MQAE39 (Figure 7.7)
See Table 7.4 below for properties.
Figure 7.7 MQAE
Figure 7.6 SPQ
Table 7.4 - Chloride Indicators
KSV a
Catalog
Number
MW
(g/mol)
ABSmax
(nm)
EXmax
(nm)
EMmax
(nm)
(M-1)
SPQ
0050
281
350
350
442
118
H2O
MQAE
0051
326
350
350
460
200
H2O
Solubility
The Stern-Volkmann constant (KSV) listed is the inverse of the concentration of Cl- needed to effect 50% quenching. It is sensitive to temperature, viscosity, ionic strength,
and cellular interactions. Scientists should calibrate the indicator to the experimental sytem being used.
a
7.4 Zinc Indicators
Zinc (Zn2+) is an essential divalent cation in living systems. Zinc research is growing, especially in the context of neurotransmission,
epilepsy and Alzheimer’s disease. Zn2+ is involved or implicated in apoptosis, immune function, gene expression, DNA synthesis,
etc. TEFLabs currently offers TSQ (Figure. 7.8), TFLZn (Figure 7.9), and Zinquin (Figure 7.10). (Table 7.5 shows their properties.)
32
Figure 7.8 TSQ
Figure 7.9 TFLZn
Figure 7.10 Zinquin
Table 7.5 - Zinc Indicators
a
b
Catalog
Number
MW
ABSmax
(nm)
EXmax
(nm)
Solubility
TSQ
0010
328
334
385
MeOH
TFL-Zn (AM)
0013
450
N/Aa
N/Aa
DMSO
TFL-Zn (Free acid)
0012
378
360
498
H2O
TFL-Zn (K+ salt)
0011
416
360
498
H2O
ZINQUIN (AM)
0015
458
N/Aa
N/Aa
DMSO
ZINQUIN (Free acid)
0014
386
360
500
H2O
Once the AM ester form permeates the cell membrane, intracellular non-specific esterases hydrolyze the AM ester to yield the indicator in its Zn2+ sensitive salt form.
The free acid should be dissolved at pH > 7.
Chapter 7 - Other Indicators
33
Chapter 8
IONOPHORES
8.1 Sodium Ionophores
The two ionophores SQI-Et (Figure 8.1) and SQI-Pr (Figure 8.2) are electrogenic sodium (Na+) ionophores developed by Drs.
Tsien and Minta. These ionophores facilitate the transport of Na+ ions through lipid membranes, with the net effect of allowing
Na+ to flow from a compartment with higher Na+ concentration, through the membrane, into a compartment with lower Na+
concentrations. For example, these ionophores can be used to equilibrate intracellular and extracellular Na+ concentrations.
Figure 8.1 SQI-Et
Figure 8.2 SQI-Pr
The biophysical studies on erythrocyte “ghosts” performed by Dr. Mario Moronne at the Department of Physiology, University
of California, Berkeley40 showed that SQI-Et has a selectivity for Na+ over K+ of 100:1. The ionophores carry Na+ ions across
the cell membrane and consequently raise the cell membrane potential. (Figures 8.3 and 8.4).
Figure 8.3: Effect of SQI-Pr on membrane polarization (Em). Neutrophils were
equilibrated with 25 nM DiS-C3 in Na+-rich medium, and fluorescence was recorded.
Where indicated, 765 nM SQI-Pr was added. SQI-Pr promoted Na+ influx, which
depolarized the membrane to positive voltages (towards the equilibrium potential
of Na+), causing an increase in DiS-C3 fluorescence. Subsequent addition of 5.9 µM
gramicidin D, an ionophore that transports both Na+ and K+, drove the membrane
potential toward 0 mV, decreasing DiS-C3 fluorescence in the process.41
Figure 8.3
Figure 8.4: Increase in the fluorescence of membrane potential indicator
DiS-C3 due to depolarization by SQI-Et (top) and SQI-Pr (bottom). Red
blood cells (RBC’s) were exposed to DiS-C3, and their membranes were
subsequently depolarized by the SQI ionophores, resulting in an increase in
DiS-C3’s fluorescence. Hyperpolarization caused by valinomycin is seen as a
decrease in fluorescence.42
SQI-Et and SQI-Pr have an intrinsic fluorescence excitable by UV light. Therefore,
they are not well-suited for use with SBFI, which is a UV-excitable Na+ indicator.
These ionophores are ideally suited for use in conjunction with long-wavelength
Na+ indicators like ANG-1 and ANG-2. The properties of SQI-Et and SQI-Pr
are listed in Table 8.1.
Figure 8.4
34
Figure 8.5 Response of intracellular Asante Natrium Green 2 (ANG-2, Section 2.2 )
to Na+ influx facilitated by SQI-Pr: REF52 fibroblasts loaded with ANG-2 (through
incubation with AM ester) were maintained in Hanks’ Balanced Salt Solution (HBSS).
SQI-Pr is a Na+ ionophore that is expected to promote Na+/H+ exchange across the
cell membrane, and thus to cause Na+ influx. Upon application of 40 μM SQI-Pr, the
fluorescence of intracellularly-loaded ANG-2 increased steadily, reflecting a rise
in intracellular [Na+]. Further addition of 20 μM amphotericin B, a polyene natural
product that increases membrane permeability to all the common monovalent ions
(Na+, K+, H+ and Cl-), gave a small additional increment of indicator fluorescence, as
expected. This experiment demonstrates the utility of ANG-2 as a sodium indicator,
and the efficacy of SQI-Pr as a sodium ionophore. (Response shown is the average
from 30 cells.)
Figure 8.5
Table 8.1 - Electrogenic Sodium Ionophones
Catalog
Number
MW
(g/mol)
ABSmax
(nm)
EMmax
(nm)
Selectivity
(Na+/K+)
Effective
Concentration
SQI-Et (Na)
0070
677
345
475
90:1
40 µM
SQI-Pr (Na)
0071
733
345
475
100:1
800-900 nM
8.2 Potassium Ionophore
TEFLabs offers the K+ selective ionophore, valinomycin, to complement our potassium indicators APG-1 and APG-2
(section 3.2).
8.3 Calcium Ionophores
The calcium ionophore A-23187 and its brominated derivative, 4-Bromo-A-23187, are used to facilitate transport of
extracellular Ca2+ into cells. These ionophores also facilitate transport of some other divalent cations, such as manganese
(Mn2+). A-23187 has an intrinsic fluorescence excitable by UV light, making it less useful with UV-excitable Ca2+ indicators,
e.g., Fura-2, but it is still useful for long-wavelength Ca2+ indicators, e.g., Fluo-2. 4-Bromo-A-23187 is nonfluorescent and is thus
compatible with all Ca2+ indicators.
8.4 Experimental Methods
Methods for Figure 8.5: REF52 fibroblasts were incubated at room temperature for 60 minutes with 2 μM Asante Natrium
Green 2 AM ester in bicarbonate-buffered Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine
serum, under an atmosphere of 5% CO2/95% O2; the medium also contained 0.0075% (w/v) of the nonionic surfactant,
Pluoronic F-127. The cells were then transferred into HBSS. SQI-Pr was prepared as a 20 mM stock solution in DMSO;
amphotericin B was prepared as a 50 mM solution in DMSO. Appropriate volumes of the stock solutions were added to the
HBSS bathing the cells to achieve the desired final concentrations used in the experiment. Indicator fluorescence was excited at
488 nm; fluorescence images were acquired with a cooled CCD camera. [J Kao, Univ. of Maryland Medical School]
Chapter 8 - Sodium Ionophores
35
Chapter 9
STANDARD FLUOROPHORES FOR LABELING AND OTHER BIOLOGICAL APPLICATIONS
TEFLABS offers standard fluorophores for labeling, staining, genetic analysis, FRET components, etcetera. These dyes are
derived for various applications and they include the following general classes: 1) Fluoresceins, 2) Carboxyfluoresceins, and
3) Rhodamines. Table 9.1 lists the properties of these fluorophores.
Table 9.1
ε
Catalog
Number
MW
(g/mol)
ABSmax
(nm)
(cm-1M-1)
EXmax
(nm)
EMmax
(nm)
Solubility
Fluorescein
0300
332
490
88000
490
514
H20
Fluorescein Diacetate
0301
416
0280
0290
0302
0303
0304
0305
0306
0307
0308
0309
0310
0311
0312
0313
0500
0501
0502
401
485
376
376
376
460
460
460
473
473
473
557
557
557
445
445
445
N/A*
95000
N/A*
504
N/A*
529
DMSO
Dichlorofluorescein
Dichlorofluorescein Diacetate
5(6) FAM
5 FAM
6 FAM
5(6) FAM Diacetate
5 FAM Diacetate
6 FAM Diacetate
5(6) FAM SE
5 FAM SE
6 FAM SE
5(6) FAM SE Diacetate
5 FAM SE Diacetate
6 FAM SE Diacetate
5(6) Carboxy Dichlorofluorescein
5 Carboxy Dichlorofluorescein
6 Carboxy Dichlorofluorescein
N/A*
504
N/A*
492
492
492
N/A*
78000
79000
8100
N/A*
492
492
492
N/A*
517
518
515
N/A*
N/A*
N/A*
495
494
496
N/A*
N/A*
N/A*
74000
78000
83000
N/A*
N/A*
N/A*
495
494
496
N/A*
N/A*
N/A*
519
520
516
N/A*
N/A*
N/A*
504
504
504
N/A*
N/A*
N/A*
90000
90000
90000
N/A*
N/A*
N/A*
504
504
504
N/A*
N/A*
N/A*
529
529
529
H20
DMSO
H20
H20
H20
DMSO
DMSO
DMSO
H20
H20
H20
DMSO
DMSO
DMSO
H20
H20
H20
5(6) Carboxy Dichlorofluorescein Diacetate
0503
529
N/A*
N/A*
N/A*
N/A*
DMSO
5 Carboxy Dichlorofluorescein Diacetate
0504
529
N/A*
N/A*
N/A*
N/A*
DMSO
6 Carboxy Dichlorofluorescein Diacetate
0505
529
0506
0507
0508
542
542
542
N/A*
90000
90000
90000
N/A*
504
504
504
N/A*
529
529
529
DMSO
5(6) Carboxy Dichlorofluorescein SE
5 Carboxy Dichlorofluorescein SE
6 Carboxy Dichlorofluorescein SE
N/A*
504
504
504
5(6) Carboxy Dichlorofluorescein SE Diacetate
0509
626
N/A*
N/A*
N/A*
N/A*
5 Carboxy Dichlorofluorescein SE Diacetate
0510
626
*
*
*
N/A
*
DMSO
DMSO
6 Carboxy Dichlorofluorescein SE Diacetate
5(6) TAMRA
5 TAMRA
6 TAMRA
0511
0314
0315
0316
626
430
430
430
*
N/A*
565
568
564
N/A
*
N/A
540
542
540
N/A
*
N/A
95000
91000
103000
N/A
N/A
540
542
542
H20
H20
H20
DMSO
DMSO
DMSO
DMSO
The acetates quench fluorescence and render the molecule membrane permeant. Non-specific intracellular esterases cleave the acetates to yield the membraneimpermeant fluorophore.
*
36
Chapter 10
MISCELLANEOUS PRODUCTS
10.1 Viability and Cytotoxicity Assay Products
In addition to BCECF and our fluorescein products, TEFLabs offers cell-permeant Calcein (AM) for viability and toxicity assays.
Calcein is also available as a non-fixable polar tracer. Our current list of these assay reagents is shown in Table 10.1.
Table 10.1 Reagents for Viability and Toxicity Assays
Catalog
Number
MW
(g/mol)
ABSmax
EXmax
Emmax
Solubility
BCECF (AM)
0061
821
507a,b
N/Ac
N/Ac
DMSO
Calcein (AM)
0066
995
494a,b
N/Ac
N/Ac
DMSO
Calcein FREE ACID
0067
623
494
494
517
H2Od
5(6) FAM Diacetate
0305
460
492a,b
N/Ac
N/Ac
DMSO
5(6) Carboxy DCFDA
0503
531
504a,b
N/Ac
N/Ac
DMSO
AM esters and acetates quench fluorescence and render the molecule membrane permeant. Non-specific intracellular esterases cleave the esters and acetates to
yield the membrane-impermeant fluorophore.
b
Hydrolysis of AM esters and/or acetates in basic aqueous solution yielded these absorbance maxima.
c
After hydrolysis of AM esters and/or acetates, excitation and emission maxima are 1) BCECF 507/531, 2) Calcein 494/517, 3) FAM 492/517, and 4) Carboxy
DCF 504/529.
a
10.2 Protein Kinase C (PKC) Indicators
TEFLabs continues to offer our protein kinase C (PKC) indicators43,44,45 developed at the University of Texas by Poenie and coworkers46. The general feedback is that Rim-1 is more stable and cell-permeant. Fim-1 in the K+ salt form is cell-impermeant;
Fim-1 diacetate is cell-permeant and thus can be used for loading the indicator into cells by incubation. Table 10.2 lists the
properties of the PKC indicators.
Table 10.2 PKC Indicators
a
Catalog
Number
MW (g/mol)
ABS max
(nm)
EX max
(nm)
EM max
(nm)
IC50
(µM)
Solubility
FIM-1 (Diacetate)
0082
843
456
N/Aa
N/Aa
0.07
DMSO
FIM-1 (K+ SALT)
0080
833
474
480
520
1.1
DMSO
RIM-1
0081
953
561
560
580
0.07
DMSO
Once the diacetate form permeates the cell membrane, intracellular non-specific esterases hydrolyze the acetates to yield the PKC-binding salt form of the indicator.
37
References
1. Non-Invasive Techniques in Cell Biology, Wiley Liss (1990)
41. Journal of Biological Chemistry 274, 26098 (1999)
2. Nature 290 527 (1981)
42. Personal Communication, Drs. Minta and Tsien, UC Berkeley (1987)
3. Biochemistry Journal 302, 5
43. Developmental Biology 167 482 (1995)
4. Methods in Neuroscience 19 340 (1994)
44. Brain Research 714 27 (1996)
5. American Journal of Physiology 271 C1325 (1996)
45. Hearing Research 94 24 (1996)
6. Journal of Biological Chemistry 260 3440-3450 (1985)
46. Journal of Biological Chemistry 268 15812 (1993)
7. The Na , K Pump, Part B, Cellular Aspects, Alan Liss, N.Y. (1988)
47. PNAS, 93 5368 1996
+
+
8. Molecular Basis of Thyroid Hormone Action, Academic Press (1983)
9. Journal of Biological Chemistry 264 19449 (1989)
10. Molecular Probes Handbook, 9th Edition, 854 (2002)
11. Journal of Biological Chemistry 264 8171 (1989)
12. Journal of Biological Chemistry 260 3440-3450 (1985)
13. Biochemistry 19 2396 (1980)
14. London et al U.S. Patent Number 5516911 (1996)
15. Biological Bulletin 183 368 (1993)
16. Developmental Biology 158 200 (1993)
17. Journal of Cellular Biology 122 387 (1993)
18. Science 275 1643 (1997)
19. British Journal of Pharmacology 120 65 (1997)
20. Biophysical Journal 69 2112 (1995)
21. Poenie & Minta U.S. Patent Number 5576433 (1996)
22. Clinical & Experimental Pharmacology & Physiology Supplement I S225 (1995)
23. Fundamental and Applied Toxicology 33 71 (1996)
24. British Journal of Pharmacology 118 1019 (1996)
25. Japanese Journal of Pharmacology 72 111 (1996)
26. Journal of Pharmacology & Experimental Therapeutics 279 830 (1996)
27. Journal of Cell Science 109 33 (1996)
28. British Journal of Pharmacology 116 3000 (1995)
29. American Journal of Physiology 269 F339 (1995)
30. Proceedings of National Academy of Sciences 93 5368 (1996)
31. Personal Communication, Dr. Isenberg, Univ. of Halle, Germany (1995)
32. Cell Calcium 19 335 (1996)
33. Biochemistry 27 4041 (1988)
34. American Journal of Physiology 256 C540 (1989)
35. Journal of Cell Biology 95 189 (1982)
36. Biophysical Chemistry 49 85 (2000)
37. Journal of Heterocyclic Chemistry 19 841 (1982)
38. Journal of Biological Chemistry 265 8179 (1987)
39. Analytical Biochemistry 178, 355 (1989)
40. Personal Communication, Dr. Moronne, UC Berkeley Physiology (1987)
38
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Line Total
Price List
FLUOROPHORES
Item
Size
Cat. No. Price
Item
Size
Cat. No. Price
DCF
DCFDA
Fluorescein
Fluorescein Diacetate
5(6) FAM
5 FAM
6 FAM
5(6) FAM Diacetate
5 FAM Diacetate
6 FAM Diacetate
5(6) FAM SE
5 FAM SE
6 FAM SE
5(6) FAM SE Diacetate
5 FAM SE Diacetate
6 FAM SE Diacetate
5(6) TAMRA
5 TAMRA
6 TAMRA
1g
1g
1g
1g
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
10 mg
10 mg
25 mg
10 mg
10 mg
100 mg
10 mg
10 mg
0280
0290
0300
0301
0302
0303
0304
0305
0306
0307
0308
0309
0310
0311
0312
0313
0314
0315
0316
5(6) Carboxy DCF
5 Carboxy DCF
6 Carboxy DCF
5(6) Carboxy DCFDA
5 Carboxy DCFDA
6 Carboxy DCFDA
5(6) Carboxy DCF SE
5 Carboxy DCF SE
6 Carboxy DCF SE
5(6) Carboxy DCFDA SE
5 Carboxy DCFDA SE
6 Carboxy DCFDA SE
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
100 mg
10 mg
10 mg
25 mg
10 mg
10 mg
0500
0501
0502
0503
0504
0505
0506
0507
0508
0509
0510
0511
$56
$65
$42
$42
$70
$70
$70
$69
$69
$69
$90
$120
$120
$106
$110
$110
$95
$80
$80
$51
$55
$55
$115
$120
$120
$90
$120
$120
$139
$149
$149
IONOPHORES
Item
Size
Cat. No. Price
Item
Size
Cat. No. Price
4-BrA-23187 (Ca)
A23187 (Ca)
1 mg
10 mg
0073
0072
SQI-Et (Na)
SQI-Pr (Na)
Valinomycin (K)
500 µg
500 µg
25 mg
0070
0071
0076
Item
Size
Cat. No. Price
FIM-1 (Diacetate)
FIM-1 (K+ salt)
RIM-1
250 µg
250 µg
250 µg
0082
0080
0081
$72
$72
$149
$149
$65
PKC
$120
$120
$140
miscellaneous
Item
Size
Cat. No. Price
Pluronic F127 20% (wt/v) in DMSO
1 ml
2510
$29
41
Price List Cont.
CALCIUM INDICATORS
High (and Medium) Affinity
Leakage Resistant Versions
Item
Size
Cat. No. Price
Item
Size
Cat. No. Price
500 µg
10 x 50 µg
3000
3010
$170
$190
Asante Calcium Red LeakRes AM
Asante Calcium Red LeakRes
AM
Asante Calcium Red LeakRes
K + Salt
500 µg
10 x 50 µg
250 µg
3150
3160
3170
$170
$190
$100
Fluo-2 LeakRes
Fluo-2 LeakRes
AM
AM
K + Salt
1 mg
20 x 50 µg
1 mg
0230
0232
0234
$130
$150
$130
AM
AM
K + Salt
1 mg
20 x 50 µg
1 mg
0108
0109
0110
$110
$130
$110
AM
AM
K + Salt
1 mg
20 x 50 µg
1 mg
0144
$110
0145
0146
$130
$110
Asante Calcium Red
Asante Calcium Red
AM
AM
Asante Calcium Red
K + Salt 250 µg
AM
Trial-2x50 µg
3020
3030
$100
$45
K + Salt Trial-2x25 µg
AM
500 µg
3040
3305
$25
$170
AM
10 x 50 µg
+
K Salt 250 µg
AM
Trial-2x50 µg
3315
$190
3325
3335
$100
$45
K + Salt Trial-2x50 µg
AM
250 µg
3345
3700
$25
*
K + Salt 250 µg
K + Salt Trial-2x25 µg
AM
1 mg
AM
20 x 50 µg
K + Salt 1 mg
3704
3710
0200
0202
$125
$30
$110
$130
0204
0214
$110
$15
0216
0220
0222
$15
$110
$130
0224
0102
0103
$110
$90
$115
0104
0105
0106
$90
$90
$115
0107
0119
0120
$90
$110
$130
0121
0114
$110
$90
0115
$45
Asante Calcium Red
Asante Calcium Red
Asante Calcium NearIR
Asante Calcium NearIR
Asante Calcium NearIR
Asante Calcium NearIR
Asante Calcium NearIR
Asante Calcium Green
Asante Calcium Green
Asante Calcium Green
Fluo-2 MedAff
Fluo-2 MedAff
Fluo-2 MedAff
Fluo-2 MedAff
Fluo-2 MedAff
Fluo-2 HighAff
Fluo-2 HighAff
Fluo-2 HighAff
Fura-2
Fura-2
Fura-2
AM
Trial-2x50 µg
K + Salt Trial-2x50 µg
AM
1 mg
AM
20 x 50 µg
K + Salt 1 mg
Indo-1
Indo-1
Indo-1
AM
1 mg
AM
20 x 50 µg
K + Salt 1 mg
AM
1 mg
AM
20 x 50 µg
K + Salt 1 mg
Rhod-2
Rhod-2
Rhod-2
Quin-2
Quin-2
AM
1 mg
AM
20 x 50 µg
K + Salt 1 mg
AM
10 mg
K + Salt 5 mg
Low Affinity Version
Asante Calcium Red (LowAff)
Asante Calcium Red (LowAff)
Asante Calcium Red (LowAff)
Asante Calcium Green (LowAff)
Asante Calcium Green (LowAff)
Asante Calcium Green (LowAff)
Fluo-2 LowAff (Fluo-2 FF)
Fluo-2 LowAff
Fluo-2 LowAff
Fura-2 LowAff (Fura-2 FF)
Fura-2 LowAff
Fura-2 LowAff
Indo-1 LowAff (Indo-1 FF)
Indo-1 LowAff
Indo-1 LowAff
Rhod-2 LowAff (Rhod-2 FF)
Rhod-2 LowAff
42
AM
AM
K+ Salt
AM
AM
K+ Salt
AM
AM
K+ Salt
AM
AM
K+ Salt
AM
AM
K+ Salt
AM
K+ Salt
500 µg
10 x 50
250 µg
500 µg
10 x 50
250 µg
1 mg
20 x 50
1 mg
1 mg
20 x 50
1 mg
1 mg
20 x 50
1 mg
1 mg
1 mg
µg
µg
µg
µg
µg
3050
3060
3070
3716
3718
3720
0240
0242
0244
0135
0136
0137
0138
0139
0140
0159
0160
$200
$220
$120
*
*
$150
$130
$150
$130
$110
$130
$110
$110
$130
$110
$110
$130
Fluo-2 LeakRes
Fura-2 LeakRes (Fura-PE3)
Fura-2 LeakRes
Fura-2 LeakRes
Indo-1 LeakRes (Indo-PE3)
Indo-1 LeakRes
Indo-1 LeakRes
Near Membrane Version
Asante Calcium Red NearMem
Asante Calcium Red NearMem
Asante Calcium Red NearMem
Fluo-2 NearMem (FFP-18)
Fluo-2 NearMem
Fluo-2 NearMem
Fura-2 NearMem
Fura-2 NearMem
Fura-2 NearMem
Indo-1 NearMem (FIP-18)
Indo-1 NearMem
Indo-1 NearMem
AM
AM
K+ Salt
AM
AM
K+ Salt
AM
AM
K+ Salt
AM
AM
K+ Salt
500 µg
10 x 50 µg
250 µg
1 mg
20 x 50 µg
1 mg
1 mg
20 x 50 µg
1 mg
1 mg
20 x 50 µg
1 mg
3200
3210
3220
0250
0252
0254
0125
0126
0111
0127
0128
0129
$170
$190
$100
$130
$150
$130
$110
$130
$110
$110
$130
$110
25 mg
100 mg
25 mg
1g
10 mg
10 mg
25 mg
100 mg
25 mg
100 mg
25 mg
50 mg
0090
0091
0100
0101
0147
0148
0161
0162
0163
0164
0122
0123
$60
$60
$98
$88
$111
$111
$225
$99
$126
$169
$159
$375
Item
Size
Cat. No. Price
MQAE
SPQ
100 mg
100 mg
0051
0050
Other Calcium Indicators
Half-BAPTA
Half-BAPTA
BAPTA
BAPTA
Bapta-FF
Bapta-FF
Dibromo BAPTA
Dibromo BAPTA
MAPTA
MAPTA
5,5' difluoro BAPTA
5,5' difluoro BAPTA
AM
K+ Salt
AM
K+ Salt
K+ Salt
AM
AM
K+ Salt
AM
K+ Salt
AM
K+ Salt
CHLORIDE INDICATORS
*Please see www.teflabs.com for availability
$99
$72
Price List Cont.
SODIUM INDICATORS
Item
Asante NaTRIUM Green -1
Asante NaTRIUM Green-1
Asante NaTRIUM Green-1
Asante NaTRIUM Green-1
Asante NaTRIUM Green-1
Asante NaTRIUM Green-1
SBFI
SBFI
SBFI
AM
AM
TMA+ Salt
AM
AM
TMA+ Salt
AM
AM
K+ Salt
Size
Cat. No. Price
Item
500 µg
10 x 50 µg
250 µg
5 x 500 µg
Trial-2x50 µg
Trial-2x25 µg
1 mg
20 x 50 µg
1 mg
3500
3510
3520
3530
3540
3550
0030
0031
0032
Asante NaTRIUM Green-2
Asante NaTRIUM Green-2
Asante NaTRIUM Green-2
Asante NaTRIUM Green-2
Asante NaTRIUM Green-2
Asante NaTRIUM Green-2
Size
Cat. No. Price
Item
500 µg
10 x 50 µg
250 µg
5 x 500 µg
Trial-2x50 µg
Trial-2x25 µg
1 mg
20 x 50 µg
1 mg
3600
3610
3620
3630
3640
3650
0020
0021
0022
Asante Potassium Green-2
Asante Potassium Green-2
Asante Potassium Green-2
Asante Potassium Green-2
Asante Potassium Green-2
Asante Potassium Green-2
Size
Cat. No. Price
Item
$250
$300
$125
$1,000
$65
$30
$285
$325
$240
AM
AM
TMA+ Salt
AM
AM
TMA+ Salt
SQI-Et (Na+ Ionophore)
SQI-Pr (Na+ Ionophore)
Size
Cat. No. Price
500 µg
10 x 50 µg
250 µg
5 x 500 µg
Trial-2x50 µg
Trial-2x25 µg
3502
3512
3522
3532
3542
3552
$250
$300
$125
$1,000
$65
$30
500 µg
500 µg
0070
0071
$149
$149
Size
Cat. No. Price
500 µg
10 x 50 µg
250 µg
5 x 500 µg
Trial-2x50 µg
Trial-2x25 µg
3602
3612
3622
3632
3642
3652
$210
$250
$120
$1,000
$65
$30
25 mg
0076
$65
POTASSIUM INDICATORS
Item
Asante Potassium Green-1
Asante Potassium Green-1
Asante Potassium Green-1
Asante Potassium Green-1
Asante Potassium Green-1
Asante Potassium Green-1
PBFI
PBFI
PBFI
AM
AM
TMA+ Salt
AM
AM
TMA+ Salt
AM
AM
K+ Salt
$210
$250
$120
$1,000
$65
$30
$270
$325
$260
AM
AM
TMA+ Salt
AM
AM
TMA+ Salt
Valinomycin (K+ Ionophore)
MAGNESIUM INDICATORS
Item
Size
Cat. No. Price
Asante Magnesium Red
AM
500 µg
3300
$200
Asante Magnesium Green
AM
500 µg
3730
$200
Asante Magnesium Red
Asante Magnesium Red
Fluo-2 Mg
Fluo-2 Mg
Fluo-2 Mg
Fura-2 Mg (Furaptra)
Fura-2 Mg
AM
K+ Salt
AM
AM
K+ Salt
AM
AM
10 x 50 µg
250 µg
1 mg
20 x 50 µg
1 mg
1 mg
20 x 50 µg
3310
3320
0260
0262
0264
0040
0041
$220
$120
$130
$150
$130
$108
$130
Asante Magnesium Green
Asante Magnesium Green
Indo - 1Mg (MagIndo)
Indo - 1Mg
Indo - 1Mg
AM
K+ Salt
AM
AM
K+ Salt
10 x 50 µg
250 µg
1 mg
20 x 50 µg
1 mg
3740
3750
0043
0045
0044
$220
$120
$108
$130
$108
Fura-2 Mg
K+ Salt
1 mg
0042
$108
Size
Cat. No. Price
Item
K+ Salt
Free Acid
5 mg
5 mg
0011
0012
$65
$65
AM
5 mg
0013
$90
TSQ
Zinquin
Zinquin
Size
Cat. No. Price
1 mg
1 mg
20 x 50 µg
0060
0061
0062
ZINC INDICATORS
Item
TFLZn
TFLZn
TFLZn
Size
Cat. No. Price
25mg
Free Acid
5 mg
0010
0014
$105
$88
AM
5 mg
0015
$120
Size
Cat. No. Price
1 mg
1 mg
0066
0067
pH INDICATORS
Item
BCECF
BCECF
BCECF
Free Acid
AM
AM
$72
$72
$105
Item
Calcein
Calcein
AM
Free Acid
$155
$139
43
Fluo 2
Fluo-2 is the brightest and
easiest to load of the Fluo
indicators.
(See Page 20)
Fluo-2, 3, 4 brightness comparison
Asante Calcium Green
Asante Calcium Green combines
exceptional brightness with the
largest dynamic range (220-fold)
of all Ca2+ indicators (See page 18).
ACG response to neuronal Ca2+ transients
The Ion Indicator Company
Visit www.teflabs.com
9415 Capitol View Drive
Austin, Texas 78747
P: 512. 280.5223
F: 512. 280.4997
E: [email protected]