Fluorescent Solutions in Cell Biology

Recent Developments in Cellular Imaging, Apoptosis, Viability and Cell Proliferation

Dana Brozban Technical Sales Specialist Imaging and Microscopy

This Presentation

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

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

SYTO SYTO SYTO SYTO SYTO

9 dye 13 dye 16 dye 25 dye 59 dye

Nucleoli
S32703 SYTO RNASelect green fluorescent cell stain

Endoplasmic Reticulum
E12353 E34250 E34251 S34200 B7449 ER-Tracker Blue-White DPX ER-Tracker Red ER-Tracker Green SelectFX Alexa Fluor 488 Endoplasmic Reticulum Labeling Kit Brefeldin A, BODIPY 558/568 conjugate *isomer 1*

ER-Tracker Family: Live Cell Endoplamic Reticulum Markers

E12353 E34251 ER-Tracker Blue-White DPX ER-Tracker Green

E34250 ER-Tracker Red

Golgi
A21270 N22651 B22650 B34400 antigolgin-97 Ab (Primary Ab or fixed Cells) NBD C6-ceramide complexed to BSA BODIPY FL C5-ceramide complexed to BSA BODIPY TR C5-ceramide complexed to BSA

Mitochondria
M7512 M7514 M7510 T3168 A6403 MitoTracker Red CMXRos MitoTracker Green FM MitoTracker Orange CMTMRos JC-1 anticytochrome oxidase subunit I

(from panel of Ox Phos Complex Primary Abs directed against Mitochondrial Enzymes)

Lysosomes
L7528 L7526 L7545 160 LysoTracker Red DND-99 LysoTracker Green DND-26 LysoSensor Yellow/Blue DND-

Peroxisomes
S34201 SelectFX Alexa Fluor 488 Peroxisome Labeling Kit

Cytoskeleton/Tubulin
T34075 A11126 TubulinTracker Green anti-tubulin mouse monoclonal IgG

TubulinTracker Green
Live BPAE cells + Oregon Green Taxol

Live BPAE cells + TubulinTracker Green

Drastically improved tubulin labeling in living cells

Cytoskeleton/Actin
A12379 R415 A12381 Alexa Fluor 488 phalloidin rhodamine phalloidin Alexa Fluor 594 phalloidin

Cytosol
C2925 C34552 C3100MP CellTracker Green CMFDA CellTracker Red CMTPX Calcein, AM

New

Qtracker Family

Three color Qtracker Cell Labeling

Plasma Membrane
V22885 V22886 V22887 F35355 Vybrant DiI cell-labeling solution Vybrant DiO cell-labeling solution Vybrant DiD cell-labeling solution FM 1-43FX *fixable analog of FM 1-43 membrane stain*

W11262 Alexa Fluor 594 wheat germ agglutinin

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

MitoSOXTM Red Oxidant Selectivity

70000

60000

Relative Fluorescence Units (580 nm)

50000

40000

30000

20000

10000

DE AN O

on ly

H2 O2

H2 O

Su pe ro xi d

Fe TC PP

SO

Su pe ro xi d

MitoSOXTM Red - Highly Selective Superoxide Detection In Mitochondria of Live Cells

Su

pe ro xi d

Pe ro xy ni

Dy e

HR P

tri te

MitoSOX Red mitochondrial superoxide indica

Buffer for 10 min 5 mM MitoSOXTM Red

10 mM FeTCPP for 10 min 5 mM MitoSOXTM Red

Xanthine oxidase pre-oxidation of 5 mM MSR

No accumulation in mitochondria

MitoSOX Red oxidation prevention by superoxide scavengers in live BPAE cells

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

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

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

Myelin Staining Protocol Comparisons

FluoroMyelin is More Efficient

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

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

One 20-minute label step three-color staining for general anatomical visualization, or use the dyes separately.
Mouse diencephalon crosssection

Three Labels in One Step

Navigatio n

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
Cytosol Nucleus

Vitality: Cell mediates active processes

Cytosol Nucleus

+ Propidium iodide

+ Carboxyfluorescein

Nonviable (Dead) Viable (Live) Vital (Live)

Nonvital (Dead)

How it works

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Viability & Vitality: Individual Reagent Examples

Viability
Permeant DNA Dyes (Live & Dead) SYTO 9 dye SYTO 13 dye SYTO 16 dye SYTO 25 dye SYTO 59 dye Impermeant DNA Dyes (Dead) SYTOX Blue SYTOX Green Propidium iodide (PI) 7-AAD TO-PRO-3 dye Ethidium homodimers

Vitality
Cell Function Markers
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

Live Cell Dead 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)

Calcein AM and Ethidium Homodimer-1

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 M camptothecin for 4 hours, then stained with the LIVE/DEAD Cell Vitality Assay Kit
SYTOX Green dye fluorescence
120

100

C12 resazurin alamarBlue

% fluorescence remaining in cells

75

50

25

10

20

30

40

50

60

Time (minutes)

C12 resazurin fluorescence

Increased permeability / brighter signals than alamarBlue33

Viability: Kit Examples

LIVE/DEAD Sperm Viability Kit (L7011)

SYBR 14 dye and propidium iodide

LIVE/DEAD Reduced Biohazard Cell Viability Kit #2 (L23101) 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

L34955 LIVE/DEAD Fixable Violet Dead Cell Stain Kit S34857 SYTOX Blue Dead Cell Stain

Cytoplasmic Labeling Fixable Assay Ideal for work with pathogens Also available in Red, Green, and Blue

Nucleic Acid Labeling Excites with 405nm laser Quantitative, single-step dead cell indicator Minimal Spectral Overlap

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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

No washes, extractions or long incubations Convenient Samples can be frozen Sensitive and Accurate Linear from 50 to 50,000 cells; specific for total nucleic acids An excellent alternative to 3H-thymidine No Radioactivity incorporation assays

Quick and Easy

X
New CyQuant NF No Freeze
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New: Cell Cycle Analysis

V35003 Vybrant DyeCycleTM Violet Stain

G0/G1

Identifies major phases of cell cycle -G0/G1, S, G2/M

Excellent tool for Violet laser

G2/M

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

Sku # L3224 Kit Name LIVE/DEAD Viability/ Cytotoxicity Kit LIVE/DEAD CellMediated/Cytotoxicity Kit LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 LIVE/DEAD Reduced Biohazard Cell Viability Kit #2 LIVE/DEAD Reduced Biohazard Cell Viability Kit #3 LIVE/DEAD Reduced Biohazard Cell Viability Kit #4 LIVE/DEAD Fixable Violet Dead Cell Stain Kit LIVE/DEAD Sperm Viability Kit Live Stain Calcein AM Dead Stain Ethidium Homodimer1 Propidium Iodide Excitation 488nm Fix? No

L7010

DiOC18 (3)

488nm

No

L7013

SYTO 10 dye

Reactive Red

488nm

Yes

L23101

Blue Dye dim Red Dye dim Green Dye dim Violet Dye dim SYBR 14 dye

Blue Dye bright Red Dye bright Green Dye bright Violet Dye bright Propidium Iodide

UV

Yes

L23102

488nm

Yes

L23105

488nm

Yes

L34955 New L7011

405 nm 488nm

Yes No

LIVE/DEAD lineup: pick your colors

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Apoptosis Programmed Cell Death

Ca2+ Chemical or Irradiation

Ca2+

Calpain Activation Bcl-2 dATP

O2 O2

PARP

NAD m Mitochondria EndoG AIF Free Radicals

pCasp-9

Apoptosome

APAF-1 APAF-1 Cyto pCasp-9

Cyto c c

Cyto c
EffectorCaspase-3 CaspaseCaspase-6 Caspase-7

Energy

Caspase-9

DNA Fragmentation

DNA Damage

Apoptosis

Necrosis

One result many avenues

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Timeframes within Camptothecin / Jurkat Model*

Function Reagent Relative Time

Mitochondria Activity Changes MitoTracker Red dye Membrane Potential DiOC2(3), DiIC1(5), JC-1 Transition Pore MTP assay PS Translocation Caspase Activity Metabolic Activity DNA Condensation Membrane Integrity DNA Fragmentation Increase Decrease Partial Change Annexin Caspase substrates C12 resazurin Hoechst PI, SYTOX Green dye TUNEL
ASSUMED

* Jurkat cells induced with 10 M camptothecin

Putting it into a framework

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Loss of Membrane Asymmetry: Annexin V

Ca2+ Chemical or Irradiation

Ca2+

Calpain Activation Bcl-2

O2 O2

PARP

Annexin V ConjugatesdATP Alexa Fluor 350 (346/442) pCasp-9 Cyto c APAF-1 Cyto c Apoptosome Alexa Fluor 488 (495/519) APAF-1 Cyto c pCasp-9 Fluorescein (496/519) EffectorCaspase-3 CaspaseCaspase-6 EndoG Oregon Green 488 (496/524) Caspase-7 Alexa Fluor 568 (578/603) Caspase-9 Alexa Fluor 594 (590/617) DNA Fragmentation Alexa Fluor 647 (650/668) Allophycocyanin (650/660) Biotin Vybrant Apoptosis Kits (annexin, Apoptosis viability, vitality)

NAD

Energy Mitochondria Free Radicals AIF

DNA Damage

Necrosis

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

Apoptot ic

Induced with camptothecin Dead

Viable

Apoptot ic

Detection of phosphatidyl serine translocation

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Selection Guide for Vybrant Apoptosis Assay Kits

Kit # Kit #1 Kit #2 Kit #3 Kit #4 Cat # V13240 V13241 V13242 V13243 Viable Cell Stain None None None None Apoptotic Cell Stain Alexa Fluor 488 dye -Annexin V Alexa Fluor 488 dye Annexin V FITC-Annexin V YO-PRO-1 dye (Green) Hoechst 33342 (Blue)Bright YO-PRO-1 dye Hoechst 3342 Kit #8 Kit #9 Kit #10 Kit #11 V35112 V35113 V35114 V35116 None None C12 -resauzurin (Red) MitoTracker Red dye R-Phycoerythrin-Annexin V (Orange) Dead Cell Stain SYTOX Green PI (Red) PI (Red) YO-PRO-1 dye PI (Red) Kit #5 Kit #7 V13244 V23201 Hoechst 33342 dim Hoechst 33342 dim PI (Red) YO-PRO-1 dye PI (Red) SYTOX Green dye 488nm 488nm & 633nm 488nm & 633nm 488nm UV & 488nm UV & 488nm Excitation 488nm 488nm 488nm 488nm

Allophycocyanin-Annexin V SYTOX Green dye (Far Red) Allophycocyanin-Annexin V SYTOX Green dye (Far Red) Alexa Fluor 488 dye Annexin V None

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MitoProbe Reagents: Detecting Mitochondrial Changes

Ca2+ Chemical or Irradiation

Ca2+

Calpain Activation Bcl-2 dATP

O2 O2

PARP

NAD m Mitochondria EndoG AIF Free Radicals

pCasp-9

Apoptosome

APAF-1 APAF-1 Cyto pCasp-9

Cyto c c

Cyto c
EffectorCaspase-3 CaspaseCaspase-6 Caspase-7

Energy

Caspase-9

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

DNA Damage

Necrosis

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

JC-1 red fluorescence

Jurkat cells + 2 M JC-1

+ 50 M CCCP Decrease of red JC-1 signal in apoptotic cells

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

Decrease of red and green DiOC2(3) signals in apoptotic cells

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

Events

+50 M CCCP

Untreate d Decrease of far red DiIC1(5) fluorescence

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 A B C

Whole cell fluorescence Mitochondrial fluorescence Diminished fluorescence

Calcein Alone

Calcein + Cobalt

BPAE cells + Calcein calcein AM / CoCl+ Cobalt 2 + Ionomycin

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

Seeing the assay with your own eyes

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Vybrant FAM Caspase Assay Kits

Ca2+ Chemical or Irradiation

Ca2+

Calpain Activation Bcl-2 dATP

O2 O2

PARP

NAD m Mitochondria EndoG Free Radicals

pCasp-9

Apoptosome

APAF-1 APAF-1 Cyto pCasp-9

Cyto c c

Cyto c
EffectorCaspase-3 CaspaseCaspase-6 Caspase-7

Energy

Caspase-9

DNA Fragmentation

AIF Vybrant FAM Caspase-3 and 7 Assay Kit (V35118) DNA Damage Vybrant FAM Caspase-8 Assay Kit (V35119)

Vybrant FAM Polycaspases Assay Kit (V35117)

Necrosis

Apoptosis

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

Dead Cells PI Positive Increase of green FAM-FLICA Fluorescence in apoptotic cells Live Cells FAM Negative Apoptotic Cells FAM Positive

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

FAM Polycaspase fluorescence

30 minutes

1 hour

2 hours

3 hours

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

Ca2+ Chemical or Irradiation

Ca2+

Calpain Activation Bcl-2 dATP

O2 O2

PARP

NAD m Mitochondria EndoG AIF Free Radicals

pCasp-9

Apoptosome

APAF-1 APAF-1 Cyto pCasp-9

Cyto c c

Cyto c
EffectorCaspase-3 CaspaseCaspase-6 Caspase-7

Energy

Caspase-9

DNA Fragmentation Vybrant Apoptosis Assay Kit #10 (V35114)

DNA Damage

Vybrant Apoptosis Assay Kit #5 (V13244) Apoptosis APO-BrdU TUNEL Assay Kit (A23210)

Necrosis

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

Reductase

Resorufin fluorescence

4 hours camptothec in

resorufin

Necrotic (peroxidetreated)

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.

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