Embed Size (px)
Citation preview
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
Dead
Live
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
Live
Dead
Propidium iodide
LIVE/DEAD®
Fixable Red stain
Before Fixation After Fixation
488 nm excitation, 610/20 filter
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
405 nm excitation, 450/20 filter
405 nm excitation, 530/30 filter
405 nm excitation, 575/25 filter
488 nm excitation, 530/30 filter
488 nm excitation, 610/20 filter
633 nm excitation, 660/20 filter
633 nm excitation, 780/60 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
SYTO
X re
d flu
ores
cenc
e
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
Num
ber o
f Cells Co
unted control
TBHP 50‐ 200µM
BPAE
CellROX® Green Reagent (green); Hoechst 33342 (blue)
Compatible with fixation
controlTBHP
TBHP + 1mM NAC
U2OS
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
dATP
CalpainActivation
Ca2+ Chemical orγ Irradiation
Energy
Free Radicals
NAD
PARPO2
O2Bcl-2
Cyto cCyto c
Cyto cAPAF-1APAF-1
pCasp-9
pCasp-9
Caspase-9
EffectorCaspase
Ca2+
↓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
Des
crip
tion
App
licat
ion
Key
Fea
ture
s
Smarter systems | Easier cell imaging | Faster results
EL4 control cells bright field image
Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013
Treated EL4 cells bright field image
Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013
Mitochondria
dATP
CalpainActivation
Ca2+ Chemical orγ Irradiation
Energy
Free Radicals
NAD
PARPO2
O2Bcl-2
Cyto cCyto c
Cyto cAPAF-1APAF-1
pCasp-9
pCasp-9
Caspase-9
EffectorCaspase
Ca2+
↓ΔΨm
AIFEndoG
DNA Fragmentation DNA Damage
Apoptosis Necrosis
Caspase-3Caspase-6Caspase-7
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
JC-1
488 nm Excitation
Carbocyanine Dyes: MitoProbe™ JC‐1 dye3T3 cells
aaaaaaaaaaaaaaaaaaaa
Untreated Jurkat
Camptothecin Treated Jurkat
JC-1 green fluorescence
JC-1 green fluorescence
JC-1
red
fluor
esce
nce
JC-1
red
fluor
esce
nce
Eve
nts
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
Mito
Trac
ker®
Red
CM
XR
os d
ye
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
dATP
CalpainActivation
Ca2+ Chemical orγ Irradiation
Energy
Free Radicals
NAD
PARPO2
O2Bcl-2
Cyto cCyto c
Cyto cAPAF-1APAF-1
pCasp-9
pCasp-9
Caspase-9
EffectorCaspase
Ca2+
↓ΔΨm
AIFEndoG
DNA Fragmentation DNA Damage
Apoptosis Necrosis
Caspase-3Caspase-6Caspase-7
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
L A
D
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
a
Dual labeling of F2N12S vs SYTOX® AADvanced™ stain ratio of 405ex with 530/30 and 603/48 ratio emission
control
apoptotic
11
1
1
Ratio fluorescence of VL3/VL2
Ratio fluorescence of VL3/VL2
orange fluorescence VL3
orange fluorescence VL3
gree
n flu
ores
cenc
e V
L2
gree
n flu
ores
cenc
e V
L2
SY
TOX
® A
AD
vanc
ed™
fluo
resc
ence
S
YTO
X®
AA
Dva
nced
™ fl
uore
scen
ce 1
Mitochondria
dATP
CalpainActivation
Ca2+ Chemical orγ Irradiation
Energy
Free Radicals
NAD
PARPO2
O2Bcl-2
Cyto cCyto c
Cyto cAPAF-1APAF-1
pCasp-9
pCasp-9
Caspase-9
EffectorCaspase
Ca2+
↓ΔΨm
AIFEndoG
DNA Fragmentation DNA Damage
Apoptosis Necrosis
Caspase-3Caspase-6Caspase-7
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
DEVD
Active Caspase-3/7 Enzyme
Non-fluorescentNo DNA binding
Bound DNA dyeDNA dye
Read
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
Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013
Treated EL4 cells labeled with CellEvent™ Caspase 3/7 Green
Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013
EL4 control cells labeled with CellEvent™ Caspase 3/7 Green and NucBlue™ LIVE
Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013
Treated EL4 cells labeled with CellEvent™ Caspase 3/7 Green and NucBlue™ LIVE
Data collected on the EVOS® FLoid ® Imaging Station during the36th Annual Research Course in Flow Cytometry held at University of New Mexico, 2013
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)
HeLa
Increased oxidative stress
Caspase 3/7 activation
HeLa
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
SYTO
X® AAD
vanced
™ Fluorescence
CellEvent® Caspase 3/7 Green Fluorescence
SYTO
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
Nea
r IR
Liv
e/D
ead
viab
ility
“viable” cells
“early” apoptotic
“late” apoptotic
Data from Bill Telford, NCI (NIH)
Mitochondria
dATP
CalpainActivation
Ca2+ Chemical orγ Irradiation
Energy
Free Radicals
NAD
PARPO2
O2Bcl-2
Cyto cCyto c
Cyto cAPAF-1APAF-1
pCasp-9
pCasp-9
Caspase-9
EffectorCaspase
Ca2+
↓ΔΨm
AIFEndoG
DNA Fragmentation DNA Damage
Apoptosis Necrosis
Caspase-3Caspase-6Caspase-7
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
Hoechst 33342 fluorescence
Prop
idiu
m io
dide
fluo
resc
ence
Prop
idiu
m io
dide
fluo
resc
ence
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
G0/G1
S
G2M
Num
ber o
f cel
ls
G0/G1
S
G2M
4N2N
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
G0G1:
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
Cou
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
Num
ber o
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
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
BrdU
EdU
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+
C C
R’N
N+
N- R”
C C
R’
N
NN R”
Triazole
Cu+
The reaction is efficient, rapid, stable and bio-orthogonal
“click” chemistry and cell proliferation
Click chemistry-based labeling and detectionCopper catalyzed azide-alkyne cycloaddition
Azide
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)
Br
Br
Br
Br
BrdU
Br
Br
Br
Br
Incoroprated BrdU is inaccessible to the BrdU antibody in dsDNA
Br
Br
Br
Br
•BrdU antibody requires DNA denaturation for detection
•Numerous protocols: acid, heat, or nuclease for DNA denaturation
Br
Br
Br
Br
Denatured DNA is required for antibody detection of BrdU
EdU (5-ethynyl-2’-deoxyuridine)
EdU
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
EdU
-Ale
xa F
luor
® 4
88 fl
uore
scen
ce
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
Num
ber o
f Cel
ls
Clic
k-iT
™ E
dUA
lexa
Flu
or®
647
fluo
resc
ence
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:
Click-iT ® Alexa Fluor ® 488 azide
Click-iT ® Alexa Fluor ® 594 azide
Click-iT ® Alexa Fluor ® 647 azide
High-Throughput Imaging (HCS) :
Click-iT ® Alexa Fluor ® 488 azide
Click-iT ® Alexa Fluor ® 594 azide
Click-iT ® Alexa Fluor ® 647 azide
Pacific Green™ dye:Expanded Options for the Violet laser
excitation max 411 nm; emission max 500 nm
Num
ber o
f Cel
ls
CD3 –Pacific Green™ Direct Conjugate
Num
ber o
f Cel
ls
CD3-biotin + Streptavidin-Pacific Green™ Secondary
Num
ber o
f Cel
ls
CD3 + Zenon®-Pacific Green™ Mouse IgG1 complex
Num
ber o
f Cel
ls
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
Share & Learn on Facebook
Thank You!
© 2013 Life Technologies Corporation All rights reserved.Research Use Only. Not for use in diagnostic procedures.
