Webinar Molecular Probes Feb 2005

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Fluorescent Solutions in Cell Biology
Recent Developments in Cellular Imaging,
Apoptosis, Viability and Cell Proliferation
Dana Brozban Technical Sales Specialist Imaging
and Microscopy
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This Presentation

• Focus on Key Molecular Probes Products and
  Applications
• The Illuminated Cell
• Apoptosis Assays, Viability and Cell
  Proliferation

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• Nucleus
• T3605 TO-PRO-3 iodide
• S34857 SYTOX Blue
• S7020 SYTOX Green
• S11368 SYTOX Orange
• D1306 DAPI
• H3570 Hoechst 33342

SYTO 9 dye SYTO 13 dye SYTO 16 dye SYTO 25
dye SYTO 59 dye
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• Nucleoli
• S32703 SYTO RNASelect green
• fluorescent cell stain

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Endoplasmic Reticulum E12353 ER-Tracker
Blue-White DPX E34250 ER-Tracker
Red E34251 ER-Tracker Green S34200 SelectFX
Alexa Fluor 488 Endoplasmic Reticulum Labeling
Kit B7449 Brefeldin A, BODIPY 558/568 conjugate
isomer 1
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ER-Tracker Family Live Cell Endoplamic
Reticulum Markers
E12353 ER-Tracker Blue-White DPX
E34250 ER-Tracker Red
E34251 ER-Tracker Green
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Golgi A21270 antigolgin-97 Ab (Primary Ab or
fixed Cells) N22651 NBD C6-ceramide complexed to
BSA B22650 BODIPY FL C5-ceramide complexed to
BSA B34400 BODIPY TR C5-ceramide complexed to BSA
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• Mitochondria
• M7512 MitoTracker Red CMXRos
• M7514 MitoTracker Green FM
• M7510 MitoTracker Orange CMTMRos
• T3168 JC-1
• A6403 anticytochrome oxidase subunit I
• (from panel of Ox Phos Complex Primary Abs
• directed against Mitochondrial Enzymes)

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Lysosomes L7528 LysoTracker Red DND-99 L7526
LysoTracker Green DND-26 L7545 LysoSensor
Yellow/Blue DND-160
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Peroxisomes S34201 SelectFX Alexa Fluor 488
Peroxisome Labeling Kit
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Cytoskeleton/Tubulin T34075 TubulinTracker
Green A11126 antia-tubulin mouse monoclonal IgG
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TubulinTracker Green
Live BPAE cells Oregon Green Taxol
Live BPAE cells TubulinTracker Green
Drastically improved tubulin labeling in living
cells
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Cytoskeleton/Actin A12379 Alexa Fluor 488
phalloidin R415 rhodamine phalloidin A12381 Alexa
Fluor 594 phalloidin
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Cytosol C2925 CellTracker Green CMFDA C34552
CellTracker Red CMTPX C3100MP Calcein,
AM New Qtracker Family
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Three color Qtracker Cell Labeling
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Plasma Membrane V22885 Vybrant DiI cell-labeling
solution V22886 Vybrant DiO cell-labeling
solution V22887 Vybrant DiD cell-labeling
solution W11262 Alexa Fluor 594 wheat germ
agglutinin F35355 FM 1-43FX fixable analog of
FM 1-43 membrane stain
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Lipid Rafts B13950 BODIPY FL C5-ganglioside
GM1 V34403 Vybrant Alexa Fluor 488 Lipid Raft
Labeling Kit V34404 Vybrant Alexa Fluor 555
Lipid Raft Labeling Kit V34405 Vybrant Alexa
Fluor 594 Lipid Raft Labeling Kit
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MitoSOXTM Red Oxidant Selectivity
MitoSOXTM Red - Highly Selective Superoxide
DetectionIn Mitochondria of Live Cells
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MitoSOX Red oxidation prevention by superoxide
scavengers in live BPAE cells
MitoSOX Red mitochondrial superoxide indicator
10 mM FeTCPP for 10 min 5 mM MitoSOXTM Red
Buffer for 10 min 5 mM MitoSOXTM Red
Xanthine oxidase pre-oxidation of 5 mM MSR
No accumulation in mitochondria
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Cell tracers membrane probes
Classic Example DiI (pronounced dye eye)

• Diffuse along membranes
• Useful in living or fixed tissues
• Long retention time (weeks)
• Generally not well retained upon fixation
• Workhorses for neural tracing

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Cell tracers dextrans and protein tracers
Classic examples fluoro-ruby and
fluoro-emerald 10,000 MW dextrans labeled with
either rhodamine (ruby) or fluorescein (emerald)

• Must be loaded by injection
• Long retention time
• Some variants are fixable
• Group includes protein tracers such as HRP and
  cholera toxin subunit B

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Cell tracers polar tracers
Classic examples Lucifer Yellow and
Sulforhodamine 101

• Similar to dextran tracers, but are low
  molecular weight
• Must be loaded by injection or patch pipette
• Some variants are fixable
• May cross gap junctions
• Also includes labeled hydrazides and biocytin

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Myelin Staining Protocol Comparisons

FluoroMyelin is More Efficient
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FluoroMyelin Green and FluoroMyelin Red

Arbor vita of mouse cerebellum sagittal
cryosection labeled with FluoroMyelin Red,
NeuroTrace Green Nissl Stain, and DAPI.
Mouse rostral mesencephalon cross-section labeled
with FluoroMyelin Green, NeuroTrace Red Nissl
Stain, and DAPI. This combination is sold as the
new BrainStain Imaging Kit.

• Single-step dyes requiring only 20 minutes label
  time

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BrainStain Kit

• The BrainStain Imaging Kit
• DAPI
• NeuroTrace Red Nissl Stain
• FluoroMyelin Green

One 20-minute label step three-color staining
for general anatomical visualization, or use
the dyes separately.
Mouse diencephalon cross-section
Three Labels in One Step
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Navigation

• Viability and vitality reagents
• LIVE/DEAD kits
• Apoptosis Reagents
• Apoptosis timeline
• Annexin V conjugates kits
• Mitochondrial reagents
• Vybrant FAM caspase assay kits
• Metabolic and nuclear changes

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Viability Vitality

• Viability
• Cell membrane forms intact barrier

• Vitality
• Cell mediates active processes

Cytosol
Cytosol
Nucleus
Nucleus
Carboxy- fluorescein
Propidium iodide
Nonviable (Dead)
Nonvital (Dead)
Viable (Live)
Vital (Live)
How it works
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Viability Vitality Individual Reagent Examples
Viability
Vitality
Permeant DNA Dyes (Live Dead)
Impermeant DNA Dyes (Dead)
Cell Function Markers

• SYTO 9 dye
• SYTO 13 dye
• SYTO 16 dye
• SYTO 25 dye
• SYTO 59 dye

• SYTOX Blue
• SYTOX Green
• Propidium iodide (PI)
• 7-AAD
• TO-PRO-3 dye
• Ethidium homodimers

• Esterase
• CFDAs
• Calcein AM
• Oxidation
• Dihydrorhodamine 123
• C12-resazurin (Vybrant Cell Metabolic Assay)

Mix match based on spectral overlap
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Vitality by Enzyme / Metabolic Function
Dead Cell
Live Cell
Calcein AM
Ethidium Homodimer-1
Cytosol
Nucleus
AM cleavage
Laser
It isnt easy being green
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Vitality LIVE/DEAD Viability / Cytotoxicity Kit
(L3224)

• Rapid assay
• Detects live and dead cells simultaneously
• The most popular viability assay kit

Vitality metabolic activity
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New LIVE/DEAD Cell Vitality Assay Kit (L34951)

• C12 resazurin SYTOX Green dye
• Metabolic activity and viability
• 488 633 nm excitation

Jurkat cells induced with 10 µM camptothecin for
4 hours, then stained with the LIVE/DEAD Cell
Vitality Assay Kit
SYTOX Green dye fluorescence
C12 resazurin fluorescence
Increased permeability / brighter signals than
alamarBlue
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Viability Kit Examples
LIVE/DEAD Reduced Biohazard Cell Viability Kit
2 (L23101)
LIVE/DEAD Sperm Viability Kit (L7011) SYBR 14
dye and propidium iodide
Healthy cells
Aged culture
LIVE/DEAD Reduced Biohazard Cell Viability Kit
2 Principal
Heat-killed cells
Viability membrane integrity
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New Cell Vitality
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New Package Sizes CellTrace Kits
C34554 CellTrace CFSE Cell Proliferation
Kit C34555 CellTrace Oregon Green SE

• New packagings in easy to use aliquots
• Track cell divisions over multiple generations
• Existing product repackaged in experiment-size
  aliquots for ease of use and maximum shelf-life
• 488 nm excitation

Initial Intensity
7th doubling
Increased divisions decreased fluorescence
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CyQUANT Cell Proliferation Assay
Quick and Easy
No washes, extractions or long incubations
Samples can be frozen Linear from 50 to 50,000
cells specific for total nucleic acids An
excellent alternative to 3H-thymidine
incorporation assays
Convenient
Sensitive and Accurate
No Radioactivity
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New Cell Cycle Analysis
V35003 Vybrant DyeCycleTM Violet Stain

• Identifies major phases of cell cycle
• -G0/G1, S, G2/M
• Excellent tool for Violet laser
• Also Available
• V35004 Vybrant DyeCycleTM Green
• V35005 Vybrant DyeCycleTM Orange

DNA Content Analysis in LIVING Cells
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Viability Vitality LIVE/DEAD Kits
LIVE/DEAD lineup pick your colors
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Apoptosis Programmed Cell Death
One result many avenues
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Timeframes within Camptothecin / Jurkat Model
Relative Time
Jurkat cells induced with 10 µM camptothecin
Putting it into a framework
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Loss of Membrane Asymmetry Annexin V

• Annexin V Conjugates
• Alexa Fluor 350 (346/442)
• Alexa Fluor 488 (495/519)
• Fluorescein (496/519)
• Oregon Green 488 (496/524)
• Alexa Fluor 568 (578/603)
• Alexa Fluor 594 (590/617)
• Alexa Fluor 647 (650/668)
• Allophycocyanin (650/660)
• Biotin
• Vybrant Apoptosis Kits (annexin, viability,
  vitality)

Probes to detect plasma membrane changes
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Loss of Membrane Asymmetry Annexin V
Jurkat cells stained with Vybrant Apoptosis
Assay Kit 2 (V13241)
Untreated cells
Dead
Viable
Apoptotic
Induced with camptothecin
Dead
Viable
Apoptotic
Detection of phosphatidyl serine translocation
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Selection Guide for Vybrant Apoptosis Assay Kits
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MitoProbe Reagents Detecting Mitochondrial
Changes

• MitoProbe JC-1 Assay Kit (M34152)
• MitoProbe DiOC2(3) Assay Kit (M34150)
• MitoProbe DiIC1(5) Assay Kit (M34151)
• MitoProbe Transition Pore Assay Kit (M34153)
• Vybrant Apoptosis Assay Kit 11 (V35116)

Mitochondria canary in the coal mine
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MitoProbe JC-1 Assay Kit (M34152)

• Mitochondrial membrane potential
• New packaging of classic dye with robust protocol
• 488 nm excitation uses all emission
• VIP Product - Widely Used

50 µM CCCP
Jurkat cells 2 µM JC-1
JC-1 red fluorescence
JC-1 green fluorescence
Classic reagent optimized for flow cytometry
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MitoProbe DiOC2(3) Assay Kit (M34150)

• Mitochondrial membrane potential
• 488 nm excitation uses all emissions
• Easier to use than JC-1 better population
  resolution
• Can be used with APC-Annexin V conjugates for
  excellent multiparameter study of Apoptosis and
  Vitality

DiOC2(3) on HeLa cells
CCCP
Easy reagent clear result
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MitoProbe DiIC1(5) Assay Kit (M34151)

• Mitochondrial membrane potential
• 633 nm excitation allows use of 488 nm
  emissions for other reagents

DiIC1(5) on MRC5 cells
Jurkat cells stained with 50 nM DiIC1(5) 633 nm
Excitation
50 µM CCCP
Untreated
Events
DiIC1(5) fluorescence
Detection of mitochondrial changes with a 633nm
laser
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MitoProbe Transition Pore Assay Kit (M34153)

• New assay - early mitochondrial membrane event
• Direct detection of the mitochondrial
  permeability transition pore (MPTP)
• 488 nm excitation green emission use of
  orange/red channels for other reagents

D
E
A
B
C
Whole cell fluorescence
Mitochondrial fluorescence
Diminished fluorescence
BPAE cells calcein AM / CoCl2
F
G
H
Calcein Cobalt
Calcein Cobalt Ionomycin
Calcein Alone
Calcein fluorescence
Calcein fluorescence
Calcein fluorescence
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MitoProbe Transition Pore Assay Kit (M34153)
BPAE cells labeled with calcein AM, MitoTracker
Red dye, and Hoechst 33342
A
B
D
C
Seeing the assay with your own eyes
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Vybrant FAM Caspase Assay Kits

• Vybrant FAM Caspase-3 and 7 Assay Kit (V35118)
• Vybrant FAM Caspase-8 Assay Kit (V35119)
• Vybrant FAM Polycaspases Assay Kit (V35117)

Probes to detect caspase activation
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Vybrant FAM Caspase Assay Kits FLICA Function
Detection of active caspases in apoptotic cells
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Vybrant FAM Caspase Assay Kits

• Vybrant FAM Polycaspases Assay Kit (V35117)
• Vybrant FAM Caspase-8 Assay Kit (V35119)
• Vybrant FAM Caspase-3 and 7 Assay Kit (V35118)

Propidium Iodine Fluorescence

FAM Fluorescence
Reliable detection of active caspases
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Example of multiparameter analysis

• Easily combine studies of caspase activation with
  membrane asymmetry
• FAM-polycaspase Green Channel
• Propidium Iodine Red Channel
• APC-Annexin V Far Red Channel

Live cells, annexin V-positive, PI-positive
2 hours
3 hours
FAM Polycaspase fluorescence
APC annexin V fluorescence

• Note the shift in APC fluorescence (Green arrow)
  prior to shift in FAM fluorescence (Blue arrows)
• Allows study of timing of apoptoic events (I.e.
  membrane asymmetry prior to caspase activation)

Multiparameter caspase activity membrane
assymetry
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Detecting Metabolic and Nuclear Changes

• Vybrant Apoptosis Assay Kit 10 (V35114)
• Vybrant Apoptosis Assay Kit 5 (V13244)
• APO-BrdU TUNEL Assay Kit (A23210)

Metabolic nuclear changes the end game
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Vybrant Apoptosis Assay Kit 10 (V35114)

• Allophycocyanin annexin V C12 resazurin
  SYTOX Green dye
• Membrane asymmetry, metabolic activity and
  membrane integrity
• 488 633 nm excitation

Jurkat cells treated as labeled 488 nm 633 nm
excitation Live cells, annexin V-positive, SYTOX
Green-positive
A
B
C12-resazurin
4 hours camptothecin
Reductase
Resorufin fluorescence
C
D
Necrotic (peroxide-treated)
resorufin
APC annexin V
SYTOX Green Dye
Reductase activity / metabolic health
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Chromatin Condensation / DNA Fragmentation

• Vybrant Apoptosis Assay Kit 5 (V13244)
• Hoechst 33342 / propidium iodide

APO-BrdUTM TUNEL Assay Kit (A23210) Alexa Fluor
488 anti-BrdU / propidium iodide
Nuclear changes the finale
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www.invitrogen.com/iProtocol
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www.invitrogen.com/iPath
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www.invitrogen.com/Antibodies
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Literature and brochures
Easy downloads from the web, or available on
request.