Organelle stains and labels
Invitrogen Corporation, Eugene OR.
Cell Structure Labeling
• Organelle Lights™
– Organelle targeted fluorescent proteins.
• Cellular Lights™
– Fluorescent proteins targeted to other
cellular structures.
• Classic Organelle Stains
– Organic dyes or dye conjugates that
selectively label organelles or cell
structures.
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Considerations for Live Cell Labeling
• Cell Loading Methods.
How to get stuff into cells.
• Challenges to contrast.
Sources of Background.
Autofluorescence.
• Multicolor Labeling.
More labels, more information.
• Photobleaching.
The eternal challenge.
• Phototoxicity.
Killing with light.
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Guidelines for Live Cell Imaging
1. Ensure the cells/tissues are incubated
under ideal conditions.
2. Use the minimum amount of reagent to
achieve staining.
3. Use the lowest level of excitation light
possible.
4. Don’t unnecessarily expose cells to
excitation light, particularly once loaded
with dyes.
5. Maximize detector sensitivity before
increasing excitation light.
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New probes for cutting edge imaging
• Organelle Lights™ and Cellular Lights ™ Targeted FP’s for dissecting cell biology.
• Premo™ line of genetically encoded biosensors.
• Intracellular Ion Indicators.
• pHrodo ™ tools for endocytosis/ phagoctyosis.
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Baculovirus mediated gene delivery……
Targeted fluorescent proteins + BacMam
Organelle LightsTM
Cellular LightsTM
PremoTM sensors
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Organelle™ Lights
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Organelle Lights™ Organelle Markers
BacMam virus delivered expression of organelle targeted FPs
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Cellular™ Lights
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Imaging cell division with Cellular Lights™
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Membrane Trafficking
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Real time imaging of cytoskeleton disruption
Delta Vision core imaging system.
Image acquisition every 20s for 30 minutes
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A snapshot of our intracellular targets…….
SV40 nuclear localization
sequence (N- or C- terminus)
Nesprin 1alpha C-terminal
transmembrane domain (aa 923-982)
Nuclear envelope
Histones
H2B
Tubulin monomer
MAP4
human Golgi-resident enzyme
N-acetylgalactosaminyltransferase-2
LAMP
Exocyst
Exoc1 (Sec3p homologue)
Signal sequence of Calreticulin
KDEL
Synapse
Synaptophysin
leader sequence of E1alpha
pyruvate dehydrogenase
Nuclear export sequence
(N- or C- terminus)
Actin monomer
Peroxisomal C-terminal
targeting sequence
Cell surface
CSF1r (colony stimulating factor
1 receptor, cell surface marker)
Endosome
Rab5
Myristoylation/palmitoylation
sequence from Lck tyrosine kinase
Organelle lights ™ and Cellular Lights™
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Organelles Stains and Dyes
• There are a wide variety of fluorescent dyes that stain specific
intracellular structures in living cells based on their chemical
nature.
• Examples include…
– Mitotrackers for mitochondria.
– Nucleic acid stains for nuclei.
– Lysotrackers and Lysosensors for acidic compartments.
– Lipophilic stains for membranes.
– ER Tracker for Endoplasmic reticulum.
– Ceramide conjugates for Golgi apparatus.
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Live Cell Organelle Labeling
Live BPAE cells incubated simultaneously with MitoTracker® Deep Red FM,
LysoTracker® Green DND-26, and Hoechst 33342
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Mitochondrial Stains
Our most popular
– M7510
– M7512
– M22425
– M22426
MitoTracker® Red Structure
MitoTracker® Orange CMTMRos
Mitotracker® RED CMXROS 578/599 Add live and fix
MitoTracker® Red 580 580/644
MitoTracker® Deep Red 633 640/662
– M7514
MitoTracker® Green FM Live and fixable, but not
membrane potential dependent thus good for Mass determination
along with M7512
Fluorogenic reduced forms, but less retained, best for live cell
– M7511
MitoTracker® Orange CM-H2TMRos
– M7513
MitoTracker® Red CM-H2XRos
All of these improve on the classics:
Rhodamine 123 (R-302), TMRM (T668), TMRE (T-669). Though these
remain popular.
Formatted for High Content Screening/ HCS
– H10295 HCS Mitochondrial Health Kit
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Mitochondrial Probes for Physiology
M36008 MitoSox™ Red Mitochondrial SuperOxide Indicator
I35103 Image-iT® LIVE Mitochondrial Transition Pore Assay
Kit for Microscopy
A Live cell Apoptosis assay.
T3168
JC-1 At low concentrations green, in healthy
mitochondria they form red J-aggregates – thus healthy mitos
are red, compromised are green.
D22421 JC-9 Same as JC-1 but base line green signal more
stable over range of depolarizations.
TMRM (T668), TMRE (T669) have been used to measure Ca
dependent depolarization of mitochondria.
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Endoplasmic Reticulum
• E34251 ER-Tracker™ Green
• E34250 ER-Tracker™ Red
– Conjugates of drug
Gilbencalmide
– Withstand fixation but not
heavy permeabilization
• D273 DiOC6(3)
– Used in live and
glutaraldehyde fixed
– Not all cell types
(concentration-dependent
staining)
– Very phototoxic.
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Golgi Apparatus
•
N1154 NBD-ceramide
•
D3521 BODIPY® FL ceramide
•
D7540 BODIPY® TR ceramide
– Live and aldehyde fixed
cells
– Not retained through
fixation and
permeabilization
BODIPY® FL Ceramide
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Lysosome
• LysoTracker
– L7525
– L12490
– L7526
– L7528
– L12491
LysoTracker®
LysoTracker®
LysoTracker®
LysoTracker®
LysoTracker®
Blue DND-22
Blue-White DPX
Green DND-26
Red DND-99
Yellow HCK-123
– Live cell; not fixable
• LysoSensor
– Live cell; not fixable
– pH indicators
L7528 LysoTracker® Red DND-99
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Lysotracker Red in Live Cells
Lysotracker® Red DND-99 and Hoechst 33342
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Nucleic Acid Stains
• Nucleic acid stains are not specific for only one type of nucleic
acid. Depending on the staining conditions used some
cytoplasmic staining may be observed.
• There are a wide range of nucleic acid stains but only a select
few can enter live cells due to the plasma membrane
polarization.
• Examples
are…
D1306 DAPI
A1301 Acridine orange
H1399 Hoechst 33342
S7575 SYTO® 13
S7578 SYTO® 16
Structures not disclosed.
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Live Cell Nucleic Acid Labeling
SYTO 13 Live-Cell Nucleic Acid Stain
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Membrane Stains
•
Dialkylcarbocyanine and
dialkylaminostyryl probes
– D282 DiI,
– D275 DiO,
– D307 DiD
– T3163 FM® 1-43
– F35355 FM® 1-43 Fixable
– T3166 FM® 4-64
– F34653 FM® 4-64 Fixable
•
Fatty Acid analogs, phospholipids,
sphingolipids, etc
– Fatty acid analogs (label in the
carbon chain)
– Phospholipids labeled on the head
group.
FM 1-43
DiI
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Plasma Membrane Stains
CellMask™ Orange
CellMask™ Deep Red
Plasma Membrane Stain
Plasma Membrane Stain
(Above) Live 293 MSR cells labeled for 1 min in complete media, washed, and imaged
•Label maintained in PM for 30-60 minutes before internalization
•Formaldehyde-fixable (not shown in this slide) but not detergent resistance
•Near 100% labeling of cells after 1-10 min labeling
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Plasma Membrane
Labeling
Wheat Germ Agglutinin AlexaFluor 594
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Cellular Structure in Fixed Cells
• Principle method: Fixed Cell Immunofluorescence
– Use primary antibody to detect target
– Use conjugated secondary antibody to detect primary.
• Fluorescent dyes with intrinsic binding properties (both live
and fixed cells).
– Nucleic acid stains
– Organelle stains
– Membrane stains
• Other fluorescent conjugates
– Lectins
– Toxins
– Receptor Ligands
• Target Fluorescent Protein Expression systems.
– Organelle Lights™
– Cellular Lights™.
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Antibody Labeling
Anti-paxcillin AF488, Alexa Fluor 594 Phalloidin, DAPI
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Phalloidin conjugates for actin labeling
Alexa Fluor® 488 phalloidin
A12379
Alexa Fluor® 555 phalloidin
A34055
Alexa Fluor® 594 phalloidin
A12381
Alexa Fluor® 647 phalloidin
A22287
rhodamine phalloidin
R415
fluorescein phalloidin
F432
Texas Red®-X phalloidin
T7471
biotin-XX phalloidin
B7474
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Organelle Antibodies
Anti-Calreticulin detected with AF488 secondary, Mitotracker Red and DAPI
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Cell Surface Receptors
Alexa Fluor® 488 Transferrin T13342, Receptor detected with primary antibody
and Alexa Fluor® 555 conjugated secondary and Hoechst 33342 (H1390)
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Conjugates for receptor binding
transferrin from human serum, Alexa Fluor® 488 conjugate
T13342
transferrin from human serum, Alexa Fluor® 555 conjugate
T35352
transferrin from human serum, Alexa Fluor® 594 conjugate
T13343
transferrin from human serum, Alexa Fluor® 647 conjugate
T23366
transferrin from human serum, biotin-XX conjugate
T23363
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 488 streptavidin
(Alexa Fluor® 488 EGF complex)
E13345
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 555 streptavidin
(Alexa Fluor® 555 EGF complex)
E35350
epidermal growth factor, biotinylated, complexed to Alexa Fluor® 647 streptavidin
(Alexa Fluor® 647 EGF complex)
E35351
epidermal growth factor, biotin-XX conjugate (biotin EGF)
E3477
Toxin Conjugates
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Fixable whole cell labels: CellTracker Dyes
Cat #
C12881
C2925
C2927
C34551
C34552
CellTracker Probe
CellTracker Blue CMF2HC
CellTracker Green CMFDA
CellTracker Orange CMTMR
CellTracker Orange CMRA
CellTracker Red CMTPX
Abs * (nm)
371
492
541
548
577
Em * (nm)
464
517
565
576
602
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THANK YOU!
Questions?
[email protected]
800.955.6288
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