Novel Flow Cytometry Reagents from Molecular Probes · Novel Flow Cytometry Reagents from Molecular...

Jolene BradfordR&D Associate Director, Flow Cytometry Systems

June 2013

Novel Flow Cytometry Reagentsfrom Molecular Probes®

Research Use Only. Not for use in diagnostic procedures.

Are my cells performing basic functions?

Cell Health, Stress, & Death

• Cell viability & vitality

•Oxidative stress/damage•Apoptosis •Autophagy

•Cell morphology•Cell motility•Endocytosis/Phagocytosis •Transcription•Protein/gene expression •Signal transduction•Ion homeostasis

•Cell proliferation•Cell cycle

Are my cells alive?

Are my cells dividing and proliferating?

Are my cells healthy or stressed?

Are my cells performing basic functions?

Cell Health, Stress, & Death

• Cell viability & vitality

•Oxidative stress/damage•Apoptosis •Autophagy

•Cell morphology•Cell motility•Endocytosis/Phagocytosis •Transcription•Protein/gene expression •Signal transduction•Ion homeostasis

•Cell proliferation•Cell cycle

Are my cells alive?

Are my cells dividing and proliferating?

Are my cells healthy or stressed?

Viability & Vitality

Live Dead

Proliferation Membrane Potential

Enzyme Activity

Membrane Integrity

Viability detection – Easy additionsTypically measures membrane integrity− Traditional reagents (Cell‐impermeant DNA dyes)− Fixable reagents (LIVE/DEAD® Fixable dyes—also called

amine reactive dyes)

ApoptosisAutophagy

Cells exist anywhere on a continuum between healthy and dead

BA Live CellsLive + Dead Cells

Eliminate dead cells from analysis

Viability: Impermeant nucleic acid‐binding dyesViability:

Integrity of plasma membrane

Cytosol

Nucleus

Viable (Live)

Nonviable (Dead)

+ SYTOX® RedStain

Impermeant Nucleic Acid Dyes, Flow Cytometry

Dyes which penetrate cells with a compromised cell membrane to stain nucleic acids, but do not cross the membranes of live cells

> Can be used to identify dead cells in a population> Can be used to quantitate DNA content in fixed cells

Propidium Iodide (488 nm ex) 7‐AAD (488 nm ex) SYTOX® AADvanced™ dead cell stain (488 nm ex) SYTOX® Green dead cell stain (488 nm ex)SYTOX® Orange dead cell stain (488 /532/561 ex) SYTOX® Blue dead cell stain (405 nm ex)SYTOX® Red dead cell stain (633 nm ex)

SYTOX® Dead Cell StainsFive different colors for flexibility in multicolor panels

Propidium Iodide ReadyProbes® Reagent– power and simplicity

• Ready-to-use liquid propidium iodide formulation• Rapid staining of dead cells without wash steps• Ready-to-use liquid formulation in convenient dropper bottle—no need to dilute, weigh, or pipette• Stable at room temperature—keep handy at your work station or cell culture area

EL4 cells labeled with Propidium Iodide

Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013

DeadLive

LIVE/DEAD® Fixable Dead Cell Stains

Fixable violet dead cell stain405 nm Violet Excitation (440/40 BP)

Live cells: react with the kit’s fluorescent reactive dye only on their surface to yield weakly fluorescent cells.

Cells with compromised membranes: react with the dye throughout their volume, yielding brightly stained cells.

Viability = membrane integrity

After fixation

Before fixation

Propidium iodide

LIVE/DEAD®

Fixable Red stain

Before Fixation After Fixation

488 nm excitation, 610/20 filter

Effect of fixation on dead cell dyes

Before Fixation After Fixation

LIVE/DEAD® Fixable Dead Cell Stain KitsRigorous precautions for analysis of biohazardous specimens

Fixation procedures that produce minimal distortion of their characteristics are highly advantageous

Compatible with Fix & perm proceduresAdvantages of KitReduced handling risks‐ formaldehydes are known to reduce risks of virusesSpecificity and reliability –staining pattern is similar before and after fixation.

Convenience‐Cells can be stained and fixed at various times during the experiment, and the results can be analyzed several hours later, without loss of the discrimination pattern. Compatible with Fix and Perm Procedures ‐useful for dead cell identification with intracellular targets

LIVE/DEAD® Fixable Blue stain

LIVE/DEAD® Fixable Violet stain

LIVE/DEAD® Fixable Aqua stain

LIVE/DEAD® Fixable Yellow stain

LIVE/DEAD® Fixable Green stain

LIVE/DEAD® Fixable Red stain

LIVE/DEAD® Fixable Far Red stain

LIVE/DEAD® Fixable Near IR stain

Before Fixation 18 Hours Post-Fixation Before Fixation 18 Hours Post-Fixation

UV (355 nm) excitation, 450/50 filter

LIVE/DEAD® Fixable Dead Cell Stains:amine-reactive dyes

• Impermeant DNA Dyes• Add at final step, do not wash out• Emission is broad, consider for multicolor

applications• Dead/Fixed cells can be used for

compensation control• Amine‐reactive Dyes

• Do not use protein in buffers• Live cells have dim fluorescence• Use with ‐aldehyde fixatives• Can be used without fixing cells too• ArC™ compensation beads useful

Tips & Tricks

Vitality

Functionality of metabolic or enzymatic processes

Cytosol

Nucleus

Vital (Live)

Nonvital (Dead)

+ Calcein violet

Probing enzyme activity with Calcein AM

Live and ethanol-killed bovine pulmonary artery epithelial cells (BPAEC) stained with calcein AM.

Live cells with active enzymes fluoresce bright green.

• Esterase substrates– Electrically neutral– Freely diffuse into cells– Nonspecific intracellular esterases convert to fluorescent products

• Live cells– Bright fluorescence– Retained in cells

Probing enzyme activityKit Ex max Em maxCalcein Green AM 495 nm 515 nmCalcein Blue AM 360 nm 449 nmCalcein Violet AM 400 nm 452 nmCalcein Red‐Orange AM 577 nm 590 nm

Live bovine pulmonary artery endothelial cells (BPAEC) were incubated simultaneously with calcein red-orange AM and MitoTracker® Green FM and NucBLue.

Calcein violet fluorescence

Calcein fluorescence

488 nm + 633 nm Excitation

405 nm + 633 nm Excitation

Vitality: measure of metabolic activityCalcein AM

Calcein AM- 488 nm ex-Violet 405 nm ex-Blue UV ex

405 nm Excitation

Vitality: measure of metabolic activity:C12‐Resazurin

C12-Resazurin-Blue 488 nm ex

Reactive Oxygen Species(ROS)

Why is oxidative stress important?Reactive Oxygen Species (ROS) form as a natural byproduct of normal metabolism of oxygen, important role in homeostasis and cell signaling.

Increased oxidative stress has been implicated in:

AtherosclerosisNeurodegenerative disordersDiabetes and cardiovascular diseasesCancerChronic liver diseasesLung diseases

atherosclerosis

α-Synuclein (Parkinson’s)

CellROX® Green Flow Cytometry Assay Kit*for oxidative stress detection*

Uses the FITC detection channel

CellROX® Green Reagent Fluorescence

f Cells Co

unted control

TBHP 50‐ 200µM

CellROX® Green Reagent (green); Hoechst 33342 (blue)

Compatible with fixation

controlTBHP

TBHP + 1mM NAC

CellROX® Deep Red Reagent (purple); CellMask™ Orange plasma membrane stain (orange); SYTO® Green fluorescent nuclear stain (green)

U2‐OS

CellROX® Deep Red Reagent

CellROX® Deep Red Flow Cytometry Assay Kit*for oxidative stress detection*

Uses the APC detection channel

Compatible with fixation

U2‐OS

CellROX® Orange Reagent fluorescence

control

TBHPTBHP + 1mM NAC

CellROX® Orange Flow Cytometry Assay Kit*for oxidative stress detection*

Compatible yellow or green laser excitation

Apoptosis

LIVE DEAD

Apoptosis is a type of cell death

Apoptosis

Vitality/Viability Continuum

Apo = off, away ‐ptosis = a falling

Greek, apoptosis translates to “falling off" of petals from flowers, or leaves from plants or trees.

-A carefully regulated process that is part of normal development and homeostasis.

-Dysregulation of apoptosis is implicated in disease states such as cancer, autoimmune disease and degenerative conditions.

Apoptosis (αποπτοσισ)

Mitochondria

CalpainActivation

Ca2+ Chemical orγ Irradiation

Energy

Free Radicals

PARPO2

O2Bcl-2

Cyto cCyto c

Cyto cAPAF-1APAF-1

pCasp-9

Caspase-9

EffectorCaspase

↓ΔΨm

AIFEndoG

DNA Fragmentation DNA Damage

Apoptosis Necrosis

Caspase-3Caspase-6Caspase-7

Apoptosome

Apoptosis

Function Reagent Relative Time

PS translocation,Membrane Permeability YO-YRO™1, PO-PRO™-1

Annexin V, F2N12S

MitochondriaActivity changesMembrane potentialTransition pore

MitoTracker® Red dyeDiOC2(3), DiIC1(5), JC-1MTP assay

Caspase activity Cell Event™ Capsase Green, Caspase substrates

Metabolic activity C12 resazurin, Calcein AM

* Jurkat cells induced with 10 µM camptothecin

Nuclear condensation Hoechst, DyeCycle™ Violet dye

Membrane integrity PI, SYTOX® dead cell stains

Sub G0 peak DyeCycle™ Orange dye

Relative Timeframe – Jurkat Model

DeadLive

EVOS® Imaging System

XL Core XL FLoid FL/FL Color FL AutoBasic transmitted

light digital inverted system

Advanced transmitted light digital inverted

system

Basic fluorescence system

Advanced fluorescence system

Fully automated fluorescence system

Routine cell cultureIn‐hood

applications

Versatile light microscopy needsChromogenic stains(ICC, IHC), tissue

culture

Cell Culture RequiringFluorescence

Quick view of cells, fluorescent labeling

and teaching

Advanced fluorescenceimaging

Highly configurable

Advanced automated imaging

Time lapse applications, multiwell plate scanning and image stitching/ tiling

•21” monitor•Ergonomic Design

•Advanced software•Time‐lapse

•Simplified user interface•Minimal learning curve

•Up to 5 objectives•Up to 4 fluor. Channels•Capture color images•Obj./Filter flexibility

•Time‐lapse imaging•Plate scanning

Smarter systems | Easier cell imaging | Faster results

EL4 control cells bright field image

Treated EL4 cells bright field image

Mitochondria

CalpainActivation

Energy

Free Radicals

PARPO2

O2Bcl-2

Cyto cCyto c

Cyto cAPAF-1APAF-1

pCasp-9

Caspase-9

EffectorCaspase

↓ΔΨm

AIFEndoG

Apoptosis Necrosis

Apoptosome

MitoTracker® and MitoProbe™ Assay Kits− MitoTracker® Red Dye− MitoProbe™ JC-1 Assay Kit− MitoProbe™ DiIC1(5) Assay Kit− MitoProbe™ Transition Pore Assay Kit

Apoptosis Assay Kits

MitoProbe™ Reagents: Detecting Mitochondrial Changes

JC-1 on BPAE Cells

2 μM JC-1 in Jurkat

488 nm Excitation

Carbocyanine Dyes: MitoProbe™ JC‐1 dye3T3 cells

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Untreated Jurkat

Camptothecin Treated Jurkat

JC-1 green fluorescence

DiIC1(5)

TreatedHealthy

DiIC1(5) on MRC5 cells

50 nM DiIC1(5)

633 nm Excitation

Carbocyanine Dyes: MitoProbe™ DiIC1(5) dye

Data from Bill Telford, NCI (NIH)

Carbocyanine Dyes: MitoProbe™ DiIC1(5) dye- MitoProbe™ DiIC1(5)- Caspase 3- 7-AAD dead cell stain

APC-annexin V

40 nM MitoTracker® Red CMXRos

Treated

488 nm & 633 nm Excitation

MitoTracker® Red CMXRos DyeUses the PE detection channel

Healthy

Mitochondrial Membrane Potential/Annexin V Apoptosis Kit:MitoTracker® Red and Alexa Fluor® 488 annexin V

Annexin V Annexin V

control treated

Mitochondria

CalpainActivation

Energy

Free Radicals

PARPO2

O2Bcl-2

Cyto cCyto c

Cyto cAPAF-1APAF-1

pCasp-9

Caspase-9

EffectorCaspase

↓ΔΨm

AIFEndoG

Apoptosis Necrosis

Apoptosome

Annexin V Conjugates − Alexa Fluor® 350 (346/442)− Pacific Blue™ (410/455)− Alexa Fluor® 488 (495/519)− Fluorescein (496/519)− R-phycoerythrin (496/575)− Alexa Fluor® 568 (578/603)− Alexa Fluor® 594 (590/617)− Alexa Fluor® 647 (650/668)− Allophycocyanin (650/660)− Biotin

Monomeric Cyanines- Membrane permeability

Ratiometric Membrane Asymmetry - F2N12S

Cell Impermeant Nucleic Acid Stains − 7-aminoactinomycin D (7-AAD)− Propidium Iodide (PI)− SYTOX® dead cell dyes

LIVE/DEAD® Fixable Dead Cell Stains

Loss of Membrane Asymmetry / Loss of Integrity

Loss of Membrane Asymmetry: Annexin V

Control Treated

TIPS for Annexin V assays

Calcium and Magnesium are required for annexin V binding to PS; binding is reversible, so divalent cations must be present during the entire assayAnalysis should be carried out quickly following labelingSome cells (e.g. megakarocytes, platelets, some myeloid lineage cells, microvesicles) may have large amounts of PS on their surfaceIn advanced apoptotic or necrotic cells, annexin V can label the inner membrane leafletAdherent cells removed by mechanical scraping or trypsin may flip their PS residues independent of apoptosis; careful removal is required

Monomeric Cyanine DyesMembrane Permeability/Dead Cell Apoptosis Kits with

YO‐PRO™‐1 and PO‐PRO™‐1 dyes

488 nm excitation

YO-PROTM-1 fluorescence

488 nm and 405 nm excitations

Violet Ratiometric Membrane Asymmetry Probe4'‐N,N‐diethylamino‐6‐(N,N,N‐dodecyl‐methylamino‐sulfopropyl)‐methyl‐3‐hydroxyflavone

F2N12SNormal cells exhibit an asymmetry in lipid distribution between the outer and inner cell membranes with phosphatidyl‐serine (PS) and phosphatidylethanolamine (PE) normally located on the inner leaflet of the cell membrane During apoptosis PS and PE translocate from the inner to outer leaflet of the cell membrane, changing the surface charge of the outer leaflet. This translocation facilitates recognition and elimination of these cells by macrophages. The Violet Ratiometric Membrane Asymmetry Probe, F2N12S, is a novel violet excitable dye for the detection of changes in membrane asymmetry by detecting variations in surface charge, and gives two emission bands.

F2N12S Membrane Asymmetry Probe

Ratiometric dye is self calibrating

Independent of cell size, cell concentration and instrument variation

5 minute labeling time, no wash

No special buffer required

Use with adherent cells also

Dual labeling of F2N12S vs SYTOX® AADvanced™ stain ratio of 405ex with 530/30 and 603/48 ratio emission

control

apoptotic

Ratio fluorescence of VL3/VL2

orange fluorescence VL3

™ fl

Mitochondria

CalpainActivation

Energy

Free Radicals

PARPO2

O2Bcl-2

Cyto cCyto c

Cyto cAPAF-1APAF-1

pCasp-9

Caspase-9

EffectorCaspase

↓ΔΨm

AIFEndoG

Apoptosis Necrosis

Apoptosome

Caspase Activity CellEvent™ 3/7 Caspase Green ReagentVybrant® FAM Caspase-3 and –7 Assay KitVybrant® FAM Caspase-8 Assay KitVybrant® FAM Polycaspases Assay Kit

Caspase Assay Kits

A nucleic acid dye conjugated to DEVD peptide

Fluorogenic Caspase 3/7 SubstrateActive caspase 3/7cleaves the DEVD peptide and the free nucleic acid dye binds to DNA.

ADVANTAGES: − Live cell amenable, no-wash protocol

− May be added to complete growth media

− Retained after fixation and permeabilization

− May be multiplexed with other live or fixed cell probes

Active Caspase-3/7 Enzyme

Non-fluorescentNo DNA binding

Bound DNA dyeDNA dye

Add CellEvent™

reagent

Incubate30 min

CellEvent™ Caspase 3/7 Green Reagent

CellEvent® Caspase 3/7 Green Detection Reagent

Control cells Apoptosis-induced cells

U2‐OS

Caspase 3/7 +

EL4 control cells labeled with CellEvent™ Caspase 3/7 Green

Treated EL4 cells labeled with CellEvent™ Caspase 3/7 Green

EL4 control cells labeled with CellEvent™ Caspase 3/7 Green and NucBlue™ LIVE

Treated EL4 cells labeled with CellEvent™ Caspase 3/7 Green and NucBlue™ LIVE

CellEvent™ Caspase‐3/7 Green Detection Reagentfor the detection of activated caspase 3/7

-A four amino peptide (DEVD) conjugated to a nucleic acid binding dye-Cell Permeant, intrinsically non-fluorescent-With activation of caspase-3 or caspase-7 the DEVD peptide is cleaved, enabling the dye to bind to DNA producing a green fluorescence

Monitor Cell Health using CellROX® Deep Red and CellEvent ® Caspase 3/7 Green Detection Reagents

CellROX® Deep Red Reagent (purple); CellEvent® Caspase‐3/7 Green Detection Reagent (green); Hoechst 33342 (blue)

Increased oxidative stress

Caspase 3/7 activation

CellEvent® Caspase 3/7 Green Fluorescence

Annexin V Pacific Blue

® Fluo

rescen

ceMultiplexing Apoptosis Assays:

•CellEvent® Caspase 3/7 Green Detection Reagent•Annexin V Pacific Blue™ conjugate•SYTOX® AADvanced™ Dead Cell Stain

Annexin V Pacific Blue® Fluorescence

X® AAD

vanced

™ Fluorescence

CellEvent® Caspase 3/7 Green Fluorescence

X® AAD

vanced

™ Fluorescence

0 hour 1.7 hours 2.8 hours

3.3 hours 4.0 hours 4.7 hours

5.3 hours 6.2 hours 7 hours

Mitochondrial Health and Apoptosis:TMRM with CellEvent™ Caspase 3/7 Green

Tetramethylrhodamine methyl ester

HeLa Cells treated with 0.5 µM staurosporine

Immunodetection of cleaved caspase 3 (PE conjugate)and LIVE/DEAD® Fixable Near-IR labeling

Since caspase 3 immunolabeling requires fixation and permeablization, combine it with a LIVE/DEAD® Fixable Dead cell Stain.

Cells should be labeled with the Live/Dead reagent prior to fixation.

untreated camptothecin 5 μM 16 h

PE anti-cleaved caspase 3

“viable” cells

“early” apoptotic

“late” apoptotic

Data from Bill Telford, NCI (NIH)

Mitochondria

CalpainActivation

Energy

Free Radicals

PARPO2

O2Bcl-2

Cyto cCyto c

Cyto cAPAF-1APAF-1

pCasp-9

Caspase-9

EffectorCaspase

↓ΔΨm

AIFEndoG

Apoptosis Necrosis

Apoptosome

Nuclear Condensation− Apoptosis Kits

Sub-G1 Population− DyeCycle™ Orange Dye

DNA Fragmentation− Click-iT® TUNEL Assay Kit

Detecting Nuclear Changes

UV & 488 nm Excitation 405 nm & 488 nm Excitation

Camptothecin-induced Jurkat cells

Untreated Jurkat cells

Hoechst 33342 fluorescence

Condensed Chromatin

Signal

Hoechst 33342 + propidium iodide

Nuclear Condensation

DyeCycle™ Violet dye + SYTOX® AADVanced™ stain

405 nm & 488 nm Excitation

• General drug treatments– Staurosprine/camptothecin/valinomycin– Concentration/time course varies from cell– Understand cell model

• Dead Cell Control – Good to have!– Heat Killing– Alcohol Killing (cells can be stored for years)

Developing Apoptotic Controls

3 day Hands-on Apoptosis WorkshopHeld July 16-18, $750Life Technologies Training Facility in Frederick, MDThis research methods course will focus on using the flow cytometry platform to learn about apoptosis as measured with both common and cutting-edge assays. This course is aimed at individuals with beginner to intermediate experience, and consists of a series of seminars and hands-on laboratory sessions. The seminar portion of the workshop will include a brief introduction to the basics of flow cytometry and in-depth discussions of methodologies for studying apoptosis. Laboratory sessions will include sample preparation, flow cytometer instrument operation, data collection, data analysis, and troubleshooting tips. A basic fluorescent imaging system will also be available to visualize cells.

http://www.learn.lifetechnologies.com/courses/view/id/345

Proliferation:DNA Content Cell Cycle

What is cell cycle? Cell cycle describes the progression of a cell through a cycle of division.

DNA content distribution

Dye fluorescence

A single time point measurement shows cells in different phases of the cycle, with distribution in three major phases of the cell cycle.

Under ideal staining conditions, all G1 or G2 cells are expected to be uniform in staining. However in practice, the cell populations are represented on frequency histograms with peaks of various widths.

Linear scale

Frequency Histogram showing DNA content distribution

Live Jurkat cells stained with Hoechst 33342

Frequency distribution histogram & software deconvolution

Cell‐Permeant Nucleic Acid DyesDyes which have the ability to penetrate an intact cell membrane to stain nucleic acid These dyes can be used for determining the DNA content of viable cells.Allows resolution of cell cycle information against the dynamic background of LIVING cells

− Hoechst dyes (UV ex) dsDNA(A‐T)− Vybrant® DyeCycle™ Violet stain (UV, 405 ex) dsDNA− Vybrant® DyeCycle™ Green stain (488 ex) dsDNA− Vybrant® DyeCycle™ Orange stain (488 & 532 ex) dsDNA− Vybrant® DyeCycle™ Ruby stain (488–633 ex) dsDNA

Sorting: Vybrant® DyeCycle™ Orange stain

Vybrant® DyeCycle™ Orange stain: sorting of NIH 3T3 cells

Post-sort verification of populations

3 days post sortG2M:

3 days post sort

Simple Protocols

1.1 Remove the Vybrant® DyeCycle™ Violet stain from the refrigerator and allow it to equilibrate to room temperature.1.2 Prepare flow cytometry tubes each containing 1 mL of cell suspension in complete media at a concentration of 1 × 106cells/mL. 1.3 To each tube add 1 μL of Vybrant® DyeCycle™ Violet stain and mix.1.4 Incubate at 37˚C for 30 minutes, protected from light.1.5 Analyze without washing on a flow cytometer using ~405 nm excitation and ~440 nm emission.

Technical Considerations for Live Cell Cycle

Instrument− Know your instrument (lasers/emission filters)− Know your dye (excitation/emission) and read product information− Proper maintenance and careful optical alignment− Verify instrument linearitySample Prep− Single cell suspension− Cell concentration and dye concentration− Optimize for cell type, medium or buffer used, time of incubation,

temperature of incubation. Acquisition and Analysis− Acquire sample in low flow rate with traditional hydrodynamic focusing

systems− Any collection rate works with acoustic focusing systems− Total number of cells acquired− Gating strategies and software analysis

FxCycleTM Violet

ntFxCycle™ Stains: for DNA content measurements in fixed cells

Enables multicolor experiments utilizing DNA content measurements

HL-60 promyeloblast

FxCycleTM Violet stain FxCycleTM Far Red stain

FxCycleTM Far Red FxCycleTM PI/RNase

FxCycleTM PI/RNase stain

TF-1 erythroblast

Jurkat T-Lymphoblast

Proliferation: Dye Dilution

Cell division results in equal partitioning of dye between daughter cells.

Fluorescence of daughter cells is half that of parent cell

First Generation

Second Generation

Third Generation

Fourth Generation

Brightness

f Cells

Cell Proliferation Analysis by Dye Dilution

1. Bring a vial of CellTrace™ dye to room temperature.

2. Add 20µL anhydrous DMSO to prepare a 5mM stock solution.

3. Add 1µL of stock solution to 1mL cells for a final concentration of 5µM.

4. Incubate 30 minutes.

5. Quench and wash.

6. Proceed with stimulation and analysis.

CellTrace™ (Dry) DMSO

20µL DMSO

5mM CellTrace™ in DMSO

1µL dye into 1mL cells

Incubate 30min

Quench and wash Stimulate and

analyze

CellTrace™ Experimental Protocol

Dissolve

CellTrace™ CSFE Multicolor and Gating

CellTrace™ CFSE: Occupies a Popular Channel

CellTrace™ Violet: Generational Analysis

CellTrace™ Violet Analyzed with Proliferation Modeling Software

CellTrace™ Violet Multicolor and Gating

Cellular Lights® Talin GFP + CellTrace™ Violet stain

U2OS human osteosarcoma cells transduced with Cellular Lights® Talin-GFP and stained 20 minutes at room temperature with a 5µM solution of CellTrace™ Violet in phosphate-buffered saline.

Proliferation: Click-iT® EdU

Frequency Histogram DNA content distribution

Where is the S-phase?

DNA content

A549 lung cancer cells

Etoposide treated

Nocodazole treated

Frequency HistogramDNA content distribution

G1: 21.98 % G2: 62.92 %

S: 15.10 %

G1: 1.77 % G2: 82.74 %

S: 15.49 %

3H-thymidine

Radioactive

Cannot multiplex

Requires DNA denaturation for detection with antibody

Cell cycle stains require dsDNA

No DNA denaturation required for detection

Multiplex compatible – including antibodies and stains for cell cycle analysis

Thymidine Analogs

Just what is “click” chemistry?Click chemistry describes a set of chemical reactions for

use in chemical library synthesis. However the name has stuck to one conjugation reaction in particular:

Copper catalyzed azide-alkyne cycloaddition

Azide Alkyne+

N- R”

NN R”

Triazole

The reaction is efficient, rapid, stable and bio-orthogonal

“click” chemistry and cell proliferation

Click chemistry-based labeling and detectionCopper catalyzed azide-alkyne cycloaddition

Alkyne

Triazole

+Cu(I)

Room Temp

Alexa Fluor® 488 Dye

DNA DNA

This concept can be applied to the labeling and detection of DNA, using a thymidine analog containing a terminal alkyne group and a dye-labeled azide.

BrdU (5-bromo-2’-deoxyuridine)

Incoroprated BrdU is inaccessible to the BrdU antibody in dsDNA

•BrdU antibody requires DNA denaturation for detection

•Numerous protocols: acid, heat, or nuclease for DNA denaturation

Denatured DNA is required for antibody detection of BrdU

EdU (5-ethynyl-2’-deoxyuridine)

Click-iT® EdU detectionClick labeling does notrequire DNA denaturation

Dye azide reacts with the alkyne on double stranded DNA

Click-iT® EdU cell proliferation : Flow CytometrySimplified Workflow

Click-iT® EdU follows a basic protocol ofaldehyde fixation and detergent permeabilization

Fix for 15 minutes, washPermeabilize for 30 minutes, washIncubate in click labeling mixture for 30 minutes,

washOptional: Incubate with cell cycle stain for 15-30

minutesAnalyze

Attune® Acoustic Cytometer with Click-iT ® EdUAlexa Fluor® 488 azide and FxCycle™ Violet

FxCycle™ Violet fluorescence

EdU- Alexa Fluorr® 488 fluorescence

FxCycle™ Violet fluorescence

Collected at Standard 100 µl/min

Attune® Acoustic Cytometer with Click-iT® EdUAlexa Fluor® 647 azide and propidium iodide

Click-iT® EdU Alexa Fluor® 647 fluorescence

Propidium iodide fluorescence

Click-iT™ EdU cell proliferation : Flow Cytometry

Workflow Variations

Fix for 15 minutes, wash

Permeabilization for 30 min, wash

Incubate in click labeling cocktail for 30 minutes, wash

Optional: Cell Cycle stain for DNA content 15-30 min

Analyze

LIVE/DEAD® Fixable Dead Cell stain, wash

Surface immunostaining, wash

Click label and Perm together 30 min, wash

Intracellular immunostaining, wash

LIVE/DEAD® Fixable Stains: amine reactive dyescan be used with aldehyde fixation

Live cells Live & Dead cells

Violet-fluorescent reactive dye

vs SSC

Click-iT® EdU kit can be used with the amine reactive dyes

Click‐iT™ EdU detection reagents: Flow Cytometry

Pacific Blue™ azideViolet laser

Alexa Fluor® 488 azideBlue laser

Alexa Fluor® 647 azideRed laser

Click-iT ® EdU Cell Proliferation Kits Available

Flow Cytometry:

Click-iT® Alexa Fluor ® 488 azide

Click-iT ® Pacific Blue ® azide

Click-iT ® Alexa Fluor ® 647 azide

Imaging:

High-Throughput Imaging (HCS) :

Pacific Green™ dye:Expanded Options for the Violet laser

excitation max 411 nm; emission max 500 nm

CD3 –Pacific Green™ Direct Conjugate

CD3-biotin + Streptavidin-Pacific Green™ Secondary

CD3 + Zenon®-Pacific Green™ Mouse IgG1 complex

CD3 + Goat anti-Mouse Pacific Green™ Secondary

Flow Cytometry Resource Center New Fluorescence Spectraviewer

Webinars on Flow and Imaging Cytometry

Flow cytometry mobile appCell Imaging mobile app3D Cell mobile app

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Thank You!

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