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Flow Cytometry 101: the “what, why and how” IMMU7040 - immunological Methodology February 18, 2014

Flow Cytometry 101: the “what, why and how” IMMU7040 - immunological Methodology February 18, 2014. Christine Zhang, PhD Faculty of Medicine University of Manitoba. Presentation Outline. Basic Concept of Flow Cytometry analysis and instrumentation

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Flow Cytometry 101: the “what, why and how” IMMU7040 - immunological Methodology February 18, 2014

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  1. Flow Cytometry 101: the “what, why and how”IMMU7040 - immunological Methodology February 18, 2014 Christine Zhang, PhD Faculty of Medicine University of Manitoba

  2. Presentation Outline • Basic Concept of Flow Cytometry analysis and instrumentation • Applications of Flow Cytometry in Research Analysis • Fluorescence-Activated Cell Sorting (FACS) • Our Flow Cytometry Core Facility

  3. Flow Cytometry Background What is it? Emission light Optical components Detectors/amplifiers (PMT) Excitation light Lasers Digital (computer/software) Flow chamber and fluidics  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 intensityas they travel in suspension one by one past a sensing point Mixed cells with fluorescence label

  4. Flow Cytometry Background What is it for? • Cell or particle size; cytoplasmic granularity • Cell surface antigens (immunophenotyping) • intracellular antigen (e.g. cytokine profiling) • Intracellular signalling (phospho-flow) • Cell viability/apoptosis • Cell cycle, DNA content/synthesis • Cell proliferation (BRDU and CFSE) • Intracellular Ca2+flux • Cell migration and adhesion • gene expression and transfer efficiency by reporter like GFP • mRNA expression in living cells • Protein-protein interaction by FRET • Activation sate and oxidative stress • Stem cell side population analysis • Cell sorting I am a cell… I am a virus…

  5. The Generation of Fluorescent Light • Electron 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 Jablonski Diagram of Fluorescence in 1935

  6. Variation in Ex/Em of Commencially Available Fluorophores Excitation by lasers at 355nm laser (UV) Emission Spectra of Alexa series 405nm laser (violet) 488nm laser (blue) 561nm laser (green) 633nm laser (red) Each fluorochrome has its distinct excitation and emission spectra. Check if your instrument can excite and detect your fluorochromes first

  7. Flow Cytometry: How does it work Step 1: Panel Design—find the best marker-fluorophore combo for your instrument Your Fluorophore Panel – fluorescence spectra viewer • Your Marker Panel • T cells: CD3+, CD90.2+ • Effector T cell: CD4+, CD8+ • Treg: CD25+ • B cells: CD19+, B220+ • Naïve 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+ • minimize Spillover: Choose fluorochromes excited by different lasers, avoid fluorochromes in adjacent channels • Be aware of the fluorochrome brightness index and marker expression: Bright fluorochrome – low expression marker; Dim fluorochrome – high expression marker

  8. Flow Cytometry: How does it work Step 1: Example of the Marker-Fluorochrome Panel Design Goal: characterize IL-10 and TGFβ expression in Tregs in human PBMC Key: Choose colors for low expressing-intracellular proteins for which staining is complicated first. Abundant easy-to-stain surface lineage markers last. Marker Panel CD4 CD25 Foxp3 TGFβ IL-10 CD14 Fluorescence Parameters on FACSCanto • Laser Fluorochrome: • blue (488 nm) FITC, GFP, Alexa488 • PE • PerCP, PerCP-Cy5.5 • PE-Cy7 • red (633 nm) APC, Alexa647 • APC-cy7 • Violet (405 nm) Pacific blue, Dapi, V450 • AmCyan, V500, BV510 My Panel Design Fluorochrome Choice BV510 PE-Cy7 APC PE Alexa488 eFluo450 Take-Home Message: Know your marks (e.g. expression, surface or intracellular) and fluorochromes (e.g. signal intensity, stability, etc.)

  9. Flow Cytometry: How does it work (cont’d) Step 2: Sample Preparation — fluorescence labeling proteins of your interest GFP-vector transfected cells Fluorescence-tagged Ab labeled cells Anti-protein BAb Gene A GFP Gene B RFP Gene C CFP Anti-protein A Ab Gene D YFP Protein A Protein B Remember to optimize your staining protocol: e.g. cell detachment, collagenase concentration, Antibody titration, staining buffer, suitable fix/perm solution, etc.

  10. Flow Cytometry: How does it work (cont’d) Step 2: Sample Preparation — fluorescence labeling proteins of your interest: Cell Surface Staining • Collect cells and resuspend in PBS + 1% Fetal Calf Serum/BSA • Block FcγRs • Staining cells with primary antibodies-fluorochrome • Incubate on ice for 30 minutes to allow for antibody binding • Wash, optional fixation step, resuspend, analyze Intracellular staining/Phospho-Flow • Collect Cells as above • Block FcγRs • Fix and permeabilize cells • Rehydrate cells (preferred for phospho-flow analysis) • Staining with primary antibodies-fluorochrome • Incubate on ice for 30 minutes to allow for antibody binding • Wash, resuspend, analyze

  11. Flow Cytometry: How does it work (cont’d) Step 2: Sample Preparation — Know Your Cells • Cell Preparation • adherent cells need to trypsinize prior to staining  cell surface markers may be affected • large 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 level • Auto-fluorescence: some cells are intrinsically fluorescent without any staining at different level. • Autofluorescencetends to increase in all cells after fixation and prolonged storage Macrophage: High autofluorescence DC: Low autofluorescence

  12. Flow Cytometry: How does it work (cont’d) Step 3: analysis on flow cytometer FACSCalibur 2 laser system (4 fluorescence channels) FACSCantoII 3 laser system (8 fluorescence channels) LSRII 3 laser system (13 -15 fluorescence channels)

  13. Inside the Flow Cytometer: Signal Generation Sheath Flow Sample Flow Laser Beam tube sheath tank Sample injection tube (SIT) sheath filters/fluid lines Collecting lens Flow Cell interrogation point Waste tank Flow chamber 1. Fluorescence-labeled Cells are carried to the flow chamber by liquid stream 2. Florochromes are excited by laser light beam Sheath Sample

  14. Inside the Flow Cytometer: Signal Generation 3. Emitted fluorescent light pass through a set of optical filters 4. Light signals are amplified and detected by detectors (PMT). 5. Light signals are converted to digital signals and sent to computer for analysis PMT LP filter PerCP BP filter APC-Cy7 PE SSC FITC PE-Cy7 APC PE-TR

  15. Understanding the Optical Components: Longpass Filters and Bandpass Filters 3) PMT Longpass Filter APC-Cy7 % of transmission 2) BP filter 1) LP filter Wavelength (nm) BandpassFIlter APC % of transmission 3) PMT Wavelength (nm) How to read a longpass filter and bandpass filter detection range e.g.: 502LP  wavelength longer than 502nm will be allowed to pass through e.g. 530/30 = 530nm +/- (30/2)nm = 530nm +/- 15nm = 515nm – 545nm

  16. Flow Cytometry: Parameters • Forward Scatter: measures cell or particle size • Side Scatter: measures internal complexity or granularity of the cell Example of Fluorescence Parameters on LSR-II • 3 Lasers, 16 parameters (Forward Scatter, Side Scatter, 14 fluorescent detectors) • Laser Mirror/Filter Fluorochrome • blue (488 nm) 505LP, 530/30 FITC, GFP, Alexa488 • 550LP, 575/25 PE, dsRed, Alexa546, Cy3 • 600LP, 610/20 PE-Texas Red, PI (live) • 635LP, 670/14 PE-Cy5.5, 7-AAD • 735LP, 780/60 PE-Cy7 • red (633 nm) 660/20 APC, Alexa647, Cy5 • 710LP, 730/45 Alexa700 • 755LP, 780/60 APC-cy7, APC-Alexa750 • Violet (405 nm) 450/40 Pacific blue, Dapi, V450 • 475LP, 525/50 AmCyan, CFP, V500 • 545LP, 560/20 Qdot 565 • 575LP, 585/15 Qdot 585 • 595LP, 605/12 Qdot 605, BrilliantViolet605 • 630LP, 655/8 Qdot 655

  17. Flow Cytometry can detect cells or particles with a wide range of size Size beads only (0.1 – 1.1μm) Viral particles Cell-derived microparticles Primary mouse splenocytes Granularity (side scatter) size (forward scatter)

  18. 1. 2. 3. Laser Voltage time Voltage Laser time Laser time Flow Cytometry: Signal Conversion in PMT Photon  Current  Voltage  Digital Signal • Fluorescentemissionsaredetectedas a voltage pulse fromphotomultipliertube (PMT) detectors • The area, voltageandheightofthevoltage pulse ismeasured Voltage

  19. Flow Cytometry: Data Analysis • Flow cytometry data can be plotted in several different ways: • the axes of the graphs represent fluorescence intensity data, usually plotted on a log scale • for histograms, the y axis is cell number Histograms

  20. Flow Cytometry Analysis – from Simple to Complex 1 color flow (e.g transfection efficiency or 1 color staining) Monoclonal stable cell line More Granular Dead Cells and debris Bigger Cells GFP expression Whole blood Side Scatter (SSC) GFP+: 87.5% smaller cells Live Cells Receptor A Receptor B cell count Bigger Forward Light Scatter (FSC) SSC FSC GFP (MFI) Brighter

  21. Flow Cytometry Analysis – from Simple to Complex 9 color Flow (e.g. immunophenotyping and clinical diagnosis) Double Positive Population Single PE Positive Population Negative Population CD8-PE CD4-FITC Single FITC Positive Population Wood B. 2006. Arch Pathol Lab Med. 130:680–690

  22. Be Aware of signal spill-over for large panel analysis • Fluorescence 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 fluorochromeyou use along with unstained control FITC only Before compensation unstained Compensation to remove signal spillover after compensation PE - %FITC  take out x% of the signal in PE that is due to spill-over from FITC

  23. Signal Spillover and Color Compensation • Tips on compensation: • Compensation is specific for fluorochromes, NOT for cell types  compensation values are valid for all cell types • Try 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 samples • Minimize spillover by spreading your colors of choice over different lasers and avoid adjacent channels • Manual compensation to double check uncompensated compensated over compensated under compensated Median values both = ~3.2

  24. Western Blotting vsFluorescence Microscopy vs Flow Cytometry

  25. Presentation Outline • Basic Concept of Flow Cytometry analysis and instrumentation • Applications of Flow Cytometry in Research Analysis • Fluorescence-Activated Cell Sorting (FACS) • Our Flow Cytometry Core Facility

  26. Flow Cytometry Application Immunophenotyping and Cytokine Profiling – human PBMC Workflow Maecker H. Nature Reviews Immunology 12, 191-200

  27. Immunophenotyping and Cytokine Profiling – human PBMC • Marker Panel • T cells: CD3+, CD90.2+ • Effector T cell: CD4+, CD8+ • Treg: CD25+ • B cells: CD19+, B220+ • Naïve 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+ Cytokine profiling on the desired population: e.g. IFNα, IL10, CXCR3, CCR5, TFNα Maecker H. Nature Reviews Immunology 12, 191-200

  28. Immunophenotyping and Cytokine Profiling – human PBMC 10-color analysis on moncytes subsets (viability marker, lineage markers and cytokines all in 1 tube) Exclude doublets Exclude the dead PMBCs Exclude T, B, NK CXCR3 CCR2 CD16 CD14 TNFα IL10 Cytokine profiling 3 Monocyte subsets Exclude HLA-DR neg

  29. Immunophenotyping – 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  CLL • Elevated B cell number alone is not enough to diagnose various subclasses of B-Cell Neoplasia >40% CD19+ cells in PBMC CD19 CD23 CD11c CD5 CD5 CD5 CD5 CD19 CD23 CD19 CD19 CD5 CD20 Chronic Lymphocyte Leukemia (CLL) Mantle Lymphocyte Leukemia (MCL) Hairy Cell Leukemia (HCL) CD19+CD5+CD23+ CD19+CD5-CD20+CD11c+ CD19+CD5+CD23- MaryaliceStetler-Stevenson, Flow Cytometry Unit, NIH

  30. Flow Cytometry Application Receptor Signaling Cascade by Phospho-Flow Cell Surface Staining • Collect cells • Block FcγRs • Staining cells with primary antibodies-fluorochrome • Wash, resuspend, analyze Intracellular staining/Phospho-Flow • Collect Cells • Block FcγRs • Fix and permealize cells • Rehydrate cells (preferred for phospho-flow analysis) • Staining with primary antibodies-fluorochrome • Wash, resuspend, analyze

  31. Receptor Signaling Cascade by Phospho-Flow – human PBMC Key Ingredient: good phospho-specific antibodies directly conjugated to fluorophore 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 signaling molecules) Krutzik P. 2011. Flow Cytometry Protocol; Chapter 9, Fig. 2

  32. Flow Cytometry Application DNA Content and Cell Cycle Analysis Cell Cycle Phases DNA histogram G0/G1 phase (2N) S phase (2 - 4N) Debris G2/M phase (4N) Common dyes that bind stoichimetrically to DNA: Propidium Iodide, Hoechst 33342, DAPI, 7AAD ,DRAQ5, etc Tabll A. 2011. Liver Biopsy; Chapter 7, Fig. 2

  33. Flow cytometry-based Cell Cycle Analysis in human breast cancer Normal cell (diploid) G0/G1 = 60% S = 13% G2/M = 27% Diploid tetraploid Tumor cell (aneuploidy) G0/G1 = 79.5% S = 12.7% hyperdiploid G0/G1 G2/M = 7.8% Tumor Diploid hypertetraploid Count Count G2/M S DNA CONTENT DNA CONTENT Ross J. 2003. Am J ClinPathol. 120: S72-S84

  34. Flow Cytometry Application Cell Viability and Apoptosis 31% later stage Membrane blabbing Apoptotic bodies Nucleus collapse Cell dehydration Chromatin condensation Loss of PS asymmetry Apoptosis Propidiumiodide 35% early stage 33% Viable Annexin V (phosphatidyl serine)

  35. Cell Viability and Apoptosis • Early and late apoptosis (programmed cell death) can be measured based on several different type of cellular alterations • Each type of alteration can be detected by flow 1) Activation of caspases:detected by staining with antibodies detecting cleaved caspases or 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 cytoplasmic to 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-binding dyes such as DAPI or PI 5) DNA fragmentation: TUNEL assay > TdT enzymatic incorporation of fluorescent nucleotide analogues

  36. Flow Cytometry Application Cell Proliferation and DNA Replication by BrdU or CFSE BrdU/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 BrdU count EdU low-proliferating BM cells Highly-Proliferating atrial cells Propidium Iodide Non-proliferating cells Proliferating cells

  37. Presentation Outline • Basic Concept of Flow Cytometry analysis and instrumentation • Applications of Flow Cytometry in Research Analysis • Fluorescence-Activated Cell Sorting (FACS) • Our Flow Cytometry Core Facility FACSAriaIII 3 laser system (15-16 fluorescence channels)

  38. Fluorescence-Activated Cell Sorting (FACS) • FACS:a specialized type of flow cytometry to sort a heterogeneous mixture of cell suspension • Features • Sort up to 4 populations of interests • 15 fluorescence color simultaneous on the same cell • Sort different types cells • Primary BM, PBMC, mouse splenocytes • Any types of cell lines • Large fragile cells like activated neutrophiles, lung DCs • Sticky and hard to sort cells (e.g. solid tumor cells, neuron cells) • Multi-purpose sorting • 7ml round bottom tube, 15ml conical tubes. • Tissue culture plates, 96 well PCR plates • Microscope slides including multiwellchamber slides • Single cell sorting • Different modes to maximize sort purity (99% for qPCR) or recovery (for assays requiring large number of cells • sterile sorting, sample agitation, temperature control Mixture of cells to be sorted laser PMT nozzle + New drop empty drop _ _ + _ + _ _ + _ + + _ + _ + _ _ + + + _ + v

  39. FACS cell soring pros and cons Mixture of cells to be sorted • FACS sorting • Pros: • Good for sorting very rare population or any populations of interests. • Accommodate large panel of markers (up to 15 colors) • Sort up 4 populations simultaneously • Cell sample quality check by flow cytometry before sorting. • High purity, low death rate after sorting • Cons: • Require specialized instrument and dedicated personnel • May take longer to sort a large number of cells laser PMT nozzle + New drop empty drop _ _ + _ + _ _ + _ + + _ + _ + _ _ + + + _ + v

  40. Sorting Example 1: Enrichment of Ramos Cells transiently transfected with 4 different constructs Before Sorting SHIP PD EGFP Y944F After Sorting Count GFP

  41. Sorting Example 2: sort mature and immature neutrophils from mouse BM ma neu Before Sorting After Sorting imneu SSC FSC CD11b Gr-1

  42. Our Instruments – Flow Cytometry Analyzers BD FACSCanto-II Digital Flow CytometryAnalyzer Location: Room 466, Apotex Center, University of Manitoba BannatyneCampus 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 BannatyneCampus 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 nm

  43. Our Instruments – FACS sorter BD FACSAriaIIIDigital Cell Sorter Location: Room 462, Apotex Center, University of Manitoba BannatyneCampus. 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.

  44. My Contact Christine Zhang, Ph.D. Flow Cytometry Core Facility Manager Faculty of Medicine, University of Manitoba 413 Apotex Center, 750 McDermotAve. Tel: (204) 294-0691 Email: christine.zhang@med.umanitoba.ca http://umanitobaflow.ca/ Useful Resources 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/

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