Introduction to Flow Cytometry

Flow Cytometry 101: the what, why and how

IMMU7040 - immunological Methodology February 18, 2014Christine Zhang, PhDFaculty of Medicine University of Manitoba1Presentation OutlineBasic Concept of Flow Cytometry analysis and instrumentation

Applications of Flow Cytometry in Research Analysis

Fluorescence-Activated Cell Sorting (FACS)

Our Flow Cytometry Core Facility

2Flow Cytometry Background A system that integrates electronics, fluidics, optics, laser technology and computer analysis in a single platform. Allows for simultaneous multiparametric analysis of physical/chemical/biological characteristics of cells or particles at the single-cell level by detecting fluorescence intensity as they travel in suspension one by one past a sensing pointWhat is it?

Flow chamber and fluidics Emission lightOptical componentsDetectors/amplifiers (PMT)Digital (computer/software)Excitation lightLasersMixed cells with fluorescence label3Cell or particle size; cytoplasmic granularity Cell surface antigens (immunophenotyping)intracellular antigen (e.g. cytokine profiling)Intracellular signalling (phospho-flow)Cell viability/apoptosisCell cycle, DNA content/synthesisCell proliferation (BRDU and CFSE)Intracellular Ca2+ flux Cell migration and adhesiongene expression and transfer efficiency by reporter like GFPmRNA expression in living cellsProtein-protein interaction by FRETActivation sate and oxidative stress Stem cell side population analysisCell sortingWhat is it for?Flow Cytometry Background

I am a cellI am a virus4The Generation of Fluorescent Light

Jablonski Diagram of Fluorescence in 1935Electron of a fluorochrome absorbs light energy at certain wavelength (Ex)Excited to a higher energy level S1 from ground level (S0).Stair-step vibrational relaxation in picosec Energy level returns to lower level and emit fluorescent light at certain wavelength (Em)

Different fluorochromes have distinct Ex/Em

Variation in Ex/Em of Commencially Available FluorophoresEmission Spectra of Alexa seriesExcitation by lasers at488nm laser (blue)561nm laser (green)633nm laser (red)405nm laser (violet)355nm laser (UV)Each fluorochrome has its distinct excitation and emission spectra. Check if your instrument can excite and detect your fluorochromes first

Flow Cytometry: How does it workStep 1: Panel Designfind the best marker-fluorophore combo for your instrumentYour Marker Panel

T cells: CD3+, CD90.2+ Effector T cell: CD4+, CD8+Treg: CD25+

B cells: CD19+, B220+Nave B: CD27-CD138-Plasma B: CD27+CD138+

Mono/Mac: CD14+CD16- or CD16+

NK: NK1.1+,CD56+

Neutrophils: CD11chiCD11bhiCD16hi

mDCs: Lin-MHCIIhi CD11chiCD123-pDCs: Lin-MHCII- CD11c-CD123+

Your Fluorophore Panel fluorescence spectra viewerminimize Spillover: Choose fluorochromes excited by different lasers, avoid fluorochromes in adjacent channelsBe aware of the fluorochrome brightness index and marker expression: Bright fluorochrome low expression marker; Dim fluorochrome high expression marker

7Flow Cytometry: How does it workStep 1: Example of the Marker-Fluorochrome Panel DesignGoal: characterize IL-10 and TGF expression in Tregs in human PBMCKey: Choose colors for low expressing-intracellular proteins for which staining is complicated first. Abundant easy-to-stain surface lineage markers last.My Panel DesignFluorochrome Choice

BV510

PE-Cy7

APC

PE

Alexa488

eFluo450

Fluorescence Parameters on FACSCantoLaserFluorochrome:blue (488 nm)FITC, GFP, Alexa488PEPerCP, PerCP-Cy5.5PE-Cy7red (633 nm)APC, Alexa647APC-cy7Violet (405 nm) Pacific blue, Dapi, V450AmCyan, V500, BV510

Take-Home Message: Know your marks (e.g. expression, surface or intracellular) and fluorochromes (e.g. signal intensity, stability, etc.)Marker PanelCD4

CD25Foxp3

TGFIL-10CD14Matching fluorochorme intensity, marker expression and optical configuration. Give examples.

E.g. looking at IL10 and TGFbeta production in Tregs and monocytes in PBMCs.

CD4, CD25, CD14, Foxp3, TGFbeta, IL10.Decide the color for TGFbeta and IL10 first: 1) CD14 already decided on eFluor450 due to the existing antibody in the lab. 2) TGFbeta-PE (IC stain, hard to do, the only color available,PE bright and common, no tandom dye), 3) IL10-Alexa488 (IC stain hard to do, Alexa488 more stable than FITC, bright, not tandom dye). Marker expression level and fluorochrome intensity comparable. 4) Then Foxp3-APC (IC stain hard to do, choose a bright fluorochrome in a different laser). 5) CD25-PE/Cy7 and CD14-BV510 (significant signal overlap between BV510 and eFluo450). Save it for CD4 cuz CD14 and CD4 are lineage markers expressed on difference cells, both are abundantly expressed on cells). CD25 are relatively rate, so put it in PE-Cy7 channel away from others

PE-Cy7: PE-Cy7 is a tandem fluorochrome that combines PE and a cyanine dye. PE-Cy7 conjugated reagents are as bright as PE conjugates. PE-Cy7 is particularly sensitive to photo-induced degradation, resulting in loss of fluorescence and changes in fluorescence spillover. Extreme caution must be taken to avoid light exposure and prolonged exposure to paraformaldehyde fixative. Fixed cells should be analyzed within 4 hours of fixation in paraformaldehyde or transferred to a paraformaldehyde-free buffer for overnight storage.

8Step 2: Sample Preparation fluorescence labeling proteins of your interest

GFP-vector transfected cellsFluorescence-tagged Ab labeled cells

GFPGene AProtein AProtein BAnti-protein A AbFlow Cytometry: How does it work (contd)Anti-protein B AbRFPGene BCFPGene CYFPGene DRemember to optimize your staining protocol: e.g. cell detachment, collagenase concentration, Antibody titration, staining buffer, suitable fix/perm solution, etc.

Cell Surface StainingCollect cells and resuspend in PBS + 1% Fetal Calf Serum/BSABlock FcRsStaining cells with primary antibodies-fluorochrome Incubate on ice for 30 minutes to allow for antibody bindingWash, optional fixation step, resuspend, analyze

Intracellular staining/Phospho-FlowCollect Cells as aboveBlock FcRsFix and permeabilize cellsRehydrate cells (preferred for phospho-flow analysis)Staining with primary antibodies-fluorochrome Incubate on ice for 30 minutes to allow for antibody bindingWash, resuspend, analyze

Step 2: Sample Preparation fluorescence labeling proteins of your interest:

Flow Cytometry: How does it work (contd) Cell Preparationadherent cells need to trypsinize prior to staining cell surface markers may be affectedlarge and sticky cells can be difficult to work with clumping, clogging the flow cell

Non-specific Staining:FcR binding: can be blocked with antibody against FcR (Fc BLOCK)Dead or dying cells: bind every fluorescently labeled antibody at a high levelAuto-fluorescence: some cells are intrinsically fluorescent without any staining at different level.

Autofluorescence tends to increase in all cells after fixation and prolonged storageMacrophage: High autofluorescence

DC: Low autofluorescenceStep 2: Sample Preparation Know Your Cells

Flow Cytometry: How does it work (contd)

Flow Cytometry: How does it work (contd)

Step 3: analysis on flow cytometer

LSRII 3 laser system (13 -15 fluorescence channels)FACSCantoII 3 laser system (8 fluorescence channels)FACSCalibur 2 laser system (4 fluorescence channels)121. Fluorescence-labeled Cells are carried to the flow chamber by liquid stream2. Florochromes are excited by laser light beamCollecting lensLaser BeamFlow chamberSheathSampleInside the Flow Cytometer: Signal GenerationSheath FlowSample Flowsheath tanksheath filters/fluid linesFlow CelltubeSample injection tube (SIT)interrogation pointWaste tank

3. Emitted fluorescent light pass through a set of optical filters4. Light signals are amplified and detected by detectors (PMT).5. Light signals are converted to digital signals and sent to computer for analysis

Inside the Flow Cytometer: Signal Generation

FITCPESSCPerCPPE-Cy7APC-Cy7APCPE-TRPMTBP filterLP filterUnderstanding the Optical Components: Longpass Filters and Bandpass Filters

How to read a longpass filter and bandpass filter detection rangee.g.: 502LP wavelength longer than 502nm will be allowed to pass throughe.g. 530/30 = 530nm +/- (30/2)nm = 530nm +/- 15nm = 515nm 545nm

Longpass FilterBandpass FIlterAPC3) PMT1) LP filter2) BP filterAPC-Cy73) PMTWavelength (nm)% of transmissionWavelength (nm)% of transmission

Example of Fluorescence Parameters on LSR-II3 Lasers, 16 parameters (Forward Scatter, Side Scatter, 14 fluorescent detectors)

LaserMirror/FilterFluorochrome blue (488 nm)505LP, 530/30FITC, GFP, Alexa488550LP, 575/25PE, dsRed, Alexa546, Cy3600LP, 610/20PE-Texas Red, PI (live)635LP, 670/14PE-Cy5.5, 7-AAD735LP, 780/60PE-Cy7red (633 nm) 660/20APC, Alexa647, Cy5710LP, 730/45Alexa700755LP, 780/60APC-cy7, APC-Alexa750Violet (405 nm) 450/40Pacific blue, Dapi, V450 475LP, 525/50AmCyan, CFP, V500545LP, 560/20Qdot 565575LP, 585/15Qdot 585595LP, 605/12Qdot 605, BrilliantViolet605630LP, 655/8Qdot 655

Flow Cytometry: ParametersForward Scatter: measures cell or particle size

Side Scatter: measures internal complexity or granularity of the cell

Size beads only (0.1 1.1m)Cell-derived microparticles

Primary mouse splenocytesViral particlesFlow Cytometry can detect cells or particles with a wide range of size size (forward scatter)Granularity (side scatter)Viral Particles: Dr. Coombs, U of M

Microparticle: Dr. Taylor, U of M17Fluorescent emissions are detected as a voltage pulse from photomultiplier tube (PMT) detectorsThe area, voltage and height of the voltage pulse is measuredVoltageLaserLaserLasertimetimetimeVoltageVoltage1.

2.

3.

Flow Cytometry: Signal Conversion in PMTPhoton Current Voltage Digital Signal

HistogramsFlow cytometry data can be plotted in several different ways: the axes of the graphs represent fluorescence intensity data, usually plotted on a log scalefor histograms, the y axis is cell number Flow Cytometry: Data AnalysisFlow Cytometry Analysis from Simple to Complex1 color flow (e.g transfection efficiency or 1 color staining)

GFP (MFI)cell countForward Light Scatter (FSC)Side Scatter (SSC)BiggerMore GranularBigger CellsDead Cells and debrissmaller cellsLive CellsBrighter

SSCFSCMonoclonal stable cell lineWhole bloodGFP expressionReceptor AReceptor BGFP+: 87.5%Flow Cytometry Analysis from Simple to Complex

9 color Flow (e.g. immunophenotyping and clinical diagnosis)Wood B. 2006. Arch Pathol Lab Med. 130:680690Negative PopulationSingle FITC Positive PopulationSingle PE Positive PopulationDouble Positive PopulationCD8-PECD4-FITCBe Aware of signal spill-over for large panel analysisFluorescence signal spill over = signal from one fluorochrome (e.g. FITC) being picked up by detector for another fluorochrome (e.g. PE).For compensation, use single stained controls for every fluorochrome you use along with unstained control

FITC onlyunstainedCompensation to remove signal spilloverBefore compensationafter compensationPE - %FITC take out x% of the signal in PE that is due to spill-over from FITCTips on compensation:Compensation is specific for fluorochromes, NOT for cell types compensation values are valid for all cell typesTry Compensation beads if your cell samples are precious or if your marker expression is low.Choose good markers that gives sharp clear positive peak away from negative peak lymphocytes: CD4, CD8, CD90, CD19. DC/neutrophils: Gr-1, CD11b)Expression should be equal or higher than the experimental samplesMinimize spillover by spreading your colors of choice over different lasers and avoid adjacent channelsManual compensation to double check

Signal Spillover and Color Compensation uncompensatedcompensatedunder compensatedover compensated

Median values both = ~3.2 Western Blotting vs Fluorescence Microscopy vs Flow Cytometry Western BlottingFluorescence MicroscopyFlow CytometryProssemi-quantitativeno specialized instrument detectDetect different forms of a single molecule

QualitativeSingle cell basedShow molecular/cellular expression and localization fixed and liveQuantitativeSensitiveNo photobleachingSingle cell basedfixed and liveexpression of multiple molecules on the same cellConsNot single cell basednot very sensitiveprotein denaturednot quantitativeonly detect total or surface protein levelApplicationsprotein expression, maturation, phosphorylation, ubiquitylation, localization by fractionation. protein subcellular localization; cellular location protein colocalizationinter-/intra-molecular interactionlive cell imagingsurface/total expression level of proteins.developmental biology study using multiple lineage markers

And more

Presentation OutlineBasic Concept of Flow Cytometry analysis and instrumentation

Applications of Flow Cytometry in Research Analysis

Fluorescence-Activated Cell Sorting (FACS)

Our Flow Cytometry Core Facility

26Flow Cytometry ApplicationImmunophenotyping and Cytokine Profiling human PBMC

Maecker H. Nature Reviews Immunology 12, 191-200 Workflow

Immunophenotyping and Cytokine Profiling human PBMCMarker Panel

T cells: CD3+, CD90.2+ Effector T cell: CD4+, CD8+Treg: CD25+

B cells: CD19+, B220+Nave B: CD27-CD138-Plasma B: CD27+CD138+

NK: NK1.1+,CD56+

Mono/Mac: Lin-CD14+Classical: CD16-Non-classical: CD16+

mDCs: Lin-HLA-DRhi CD11chiCD123-

pDCs: Lin-HLA-DR- CD11c-CD123+

Maecker H. Nature Reviews Immunology 12, 191-200 Cytokine profiling on the desired population:e.g. IFN, IL10, CXCR3, CCR5, TFN I can study these macrophages in their normal environment in more physiological condition vs you isolate them first before studying. So effects from the surrounding cells are preserved.28Immunophenotyping and Cytokine Profiling human PBMC10-color analysis on moncytes subsets (viability marker, lineage markers and cytokines all in 1 tube)

Exclude doubletsPMBCsExclude the deadExclude T, B, NKExclude HLA-DR neg3 Monocyte subsetsCD14CD16

IL10TNFCCR2CXCR3Cytokine profilingWere from Dr. Blake Ball, U of M, National Microbiology Lab29Immunophenotyping B Cell Leukemia Diagnosis by Flow Cytometry B-cell neoplasia (e.g. CLL) can be diagnosed by flow cytometry, WBC count and clinical history.more than 5x109 monoclonal B cells in the blood CLLElevated B cell number alone is not enough to diagnose various subclasses of B-Cell NeoplasiaMaryalice Stetler-Stevenson, Flow Cytometry Unit, NIH>40% CD19+ cells in PBMC

Chronic Lymphocyte Leukemia (CLL)Mantle Lymphocyte Leukemia (MCL)CD19+CD5+CD23+CD19+CD5+CD23-

Hairy Cell Leukemia (HCL)CD19+CD5-CD20+CD11c+

CD19CD19CD5CD23CD5CD19CD5CD23CD5CD19CD5CD20CD11c

Flow Cytometry ApplicationReceptor Signaling Cascade by Phospho-Flow Cell Surface StainingCollect cells Block FcRsStaining cells with primary antibodies-fluorochrome Wash, resuspend, analyze

Intracellular staining/Phospho-FlowCollect CellsBlock FcRsFix and permealize cellsRehydrate cells (preferred for phospho-flow analysis)Staining with primary antibodies-fluorochrome Wash, resuspend, analyze

Receptor Signaling Cascade by Phospho-Flow human PBMCKey Ingredient: good phospho-specific antibodies directly conjugated to fluorophore

Krutzik P. 2011. Flow Cytometry Protocol; Chapter 9, Fig. 2

Note: phosflow is limited only to high abundance protein that are highly phosphorylated on very unique sites that can be detected with specific antibodies (a small subset of signalingmolecules)Now using Phospho-Flow, you dont need to isolate individual cell populations out of the heterogenous mixture of samples like PMBCs before doing a WB to look at cell signaling. Youll be able to look at them all together in the same tube. This way, youre not studying them in isolation because in blood, they are not isolated. Also, you require way fewer cells than thats needed for WB.

32Flow Cytometry ApplicationDNA Content and Cell Cycle Analysis

G0/G1 phase (2N)G2/M phase (4N)S phase (2 - 4N)DebrisDNA histogramCell Cycle PhasesTabll A. 2011. Liver Biopsy; Chapter 7, Fig. 2Common dyes that bind stoichimetrically to DNA: Propidium Iodide, Hoechst 33342, DAPI, 7AAD ,DRAQ5, etc

Useful resources: http://www.icms.qmul.ac.uk/flowcytometry/uses/cellcycleanalysis/cellcycle/index.html33

Flow cytometry-based Cell Cycle Analysis in human breast cancer

DiploidNormal cell (diploid)Tumor cell (aneuploidy)tetraploidhyperdiploidhypertetraploidRoss J. 2003. Am J Clin Pathol. 120: S72-S84G0/G1 = 60% S = 13% G2/M = 27%DNA CONTENTCountDNA CONTENTCountSG2/MG0/G1TumorDiploidG0/G1 = 79.5%S = 12.7%G2/M = 7.8%http://www.nature.com/bjc/journal/v108/n4/abs/bjc201342a.html. Actual analysis paper

34Cell Viability and ApoptosisFlow Cytometry Application

ApoptosisCell dehydrationChromatin condensationLoss of PS asymmetry Membrane blabbingApoptotic bodiesNucleus collapse

Propidium iodideAnnexin V (phosphatidyl serine)31% later stage33% Viable35% early stage

Apoptosis is programmed cell death where the cell goes through a highly regulated process of dying

Characteristics are condensation of the chromatin material and blebbing of nuclear material

Often accompanied by internucleosomal degradation of dna

35Early and late apoptosis (programmed cell death) can be measured based on several different type of cellular alterationsEach type of alteration can be detected by flow

1) Activation of caspases: detected by staining with antibodies detecting cleaved caspasesor caspase substrates OR staining with fluorescently-labeled caspase inhibitors (Casp-Glow)

2) Mitochondrial dysfunction: detect changes in membrane potential with Rhodamine 123, TMRE, MitoTracker dyes or cytochrome C release using specific antibodies

3) Alterations in membrane symmetry: phosphatidyl serine translocates from cytoplasmicto extracellular side of membrane > detected by annexin V binding (note: membrane inversion also occurs during granule release in neutrophils, mast cells, etc)

4) Loss of membrane integrity: apoptotic cells become permeant to DNA-bindingdyes such as DAPI or PI 5) DNA fragmentation: TUNEL assay > TdT enzymatic incorporation of fluorescent nucleotide analogues

Cell Viability and ApoptosisCell Proliferation and DNA Replication by BrdU or CFSE Flow Cytometry ApplicationBrdU/EdU: incorporated into the newly synthesized DNA of replicating cells (during the S phase)CSFE: measure cell division as CSFE fluorescence intensity is halved within daughter cells after each cell division

Propidium IodideBrdUNon-proliferating cellsProliferating cellsEdUcount

low-proliferating BM cellsHighly-Proliferating atrial cells37Presentation Outline

FACSAriaIII 3 laser system (15-16 fluorescence channels)Basic Concept of Flow Cytometry analysis and instrumentation

Applications of Flow Cytometry in Research Analysis

Fluorescence-Activated Cell Sorting (FACS)

Our Flow Cytometry Core Facility38Fluorescence-Activated Cell Sorting (FACS)v

laserPMTMixture of cells to be sortedNew dropempty drop+++++++++________+++___FACS: a specialized type of flow cytometry to sort a heterogeneous mixture of cell suspension

FeaturesSort up to 4 populations of interests15 fluorescence color simultaneous on the same cellSort different types cellsPrimary BM, PBMC, mouse splenocytesAny types of cell linesLarge fragile cells like activated neutrophiles, lung DCsSticky and hard to sort cells (e.g. solid tumor cells, neuron cells)Multi-purpose sorting7ml round bottom tube, 15ml conical tubes.Tissue culture plates, 96 well PCR platesMicroscope slides including multiwell chamber slidesSingle cell sortingDifferent modes to maximize sort purity (99% for qPCR) or recovery (for assays requiring large number of cellssterile sorting, sample agitation, temperature control

nozzleFACS cell soring pros and consFACS sortingPros:Good for sorting very rare population or any populations of interests. Accommodate large panel of markers (up to 15 colors)Sort up 4 populations simultaneouslyCell sample quality check by flow cytometry before sorting.High purity, low death rate after sorting

Cons:Require specialized instrument and dedicated personnelMay take longer to sort a large number of cellsv

laserPMTMixture of cells to be sortedNew dropempty drop+++++++++________+++___nozzle40

Sorting Example 1: Enrichment of Ramos Cells transiently transfected with 4 different constructsBefore SortingAfter SortingGFPCountEGFPPDSHIPY944F41Sorting Example 2: sort mature and immature neutrophils from mouse BM

FSCSSCGr-1CD11bBefore SortingAfter Sortingim neuma neuDr. Soussi-Gounni, U of M42Our Instruments Flow Cytometry AnalyzersBD FACSCanto-II Digital Flow Cytometry Analyzer

Location: Room 466, Apotex Center, University of Manitoba Bannatyne Campus

3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser

10 parameters: FSC, SSC, 4 fluorescent detectors off 488 nm, 2 fluorescent detectors off 633 nm, 2 fluorescent parameters off 405 nm.

BD LSR-II Digital Flow Cytometry Analyzer Location: Room 536, Basic Medical Science Building, University of Manitoba Bannatyne Campus

3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser

16 parameters: FSC, SSC, 5 fluorescent detectors off 488 nm, 3 fluorescent detectors off 633 nm, 6 fluorescent parameters off 405 nmOur Instruments FACS sorterBD FACSAriaIII Digital Cell Sorter

Location: Room 462, Apotex Center, University of Manitoba Bannatyne Campus.

3 Lasers: 1) 488 nm blue laser; 2) 633 nm red laser; 3) 405 nm violet laser

17 parameters: Forward Scatter, Side Scatter, 6 fluorescent detectors off 488 nm, 3 fluorescent detectors off 633 nm, 6 fluorescent parameters off 405 nm

Features: sample agitation; temperature control; two-way or four-way sorting into tubes, multiwell plates or microscope slides; Aerosol Management Option (AMO); housed in a ClassII Type A2 biosafety cabinet; accommodate most cell types.

My Contact Christine Zhang, Ph.D.Flow Cytometry Core Facility Manager

Faculty of Medicine, University of Manitoba413 Apotex Center, 750 McDermot Ave.

Tel: (204) 294-0691Email: christine.zhang@med.umanitoba.cahttp://umanitobaflow.ca/

http://www.cyto.purdue.edu/flowcyt/educate.htm

http://www.lifetechnologies.com/ca/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-technical-resources.html

http://www.bdbiosciences.com/research/multicolor/spectrum_viewer/

Useful Resources