Flow Cytometry Workshop

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Flow Cytometry Workshop Insert Date Dr Gareth Howell [email protected] x37270

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Flow Cytometry Workshop. Insert Date Dr Gareth Howell [email protected] x37270. Workshop: Flow Cytometry. LBFF: Leeds Bioimaging and Flow Cytometry Facility. Workshop – Flow Cytometry: Basic concepts, applications and experimental design. Workshop: Flow Cytometry. - PowerPoint PPT Presentation

Transcript of Flow Cytometry Workshop

  • Flow Cytometry WorkshopInsert Date

    Dr Gareth [email protected]

  • LBFF: Leeds Bioimaging and Flow Cytometry FacilityWorkshop Flow Cytometry:Basic concepts, applications and experimental design

    Workshop: Flow Cytometry

  • LBFF: Flow Cytometry Facility DetailsWorkshop: Flow CytometryLocation: Garstang level 8Manager: Dr Gareth Howellhttp://www.fbs.leeds.ac.uk/facilities/flowcytometry/E: [email protected]: x37270My Office

  • Workshop: Flow CytometryBD FACSCalibur2-laser, 4 colour analyser cytometerFixed emission filter set-upBD FACSAria2-laser, 7 colour analyser and cells sorting cytometerInterchangable emission filter set-upPartec PASIIISingle laser, 4 colour analyser cytometerHBO (mercury) lampInterchangable filter set-up

  • Purpose of this workshop:

    To introduce the concepts of flow cytometry (FACS)analysisTo illustrate the role flow can play in your researchDemonstrate the capabilities of flowExperimental designTo discuss the limitations of flow

    Seminar:Introduction to flowApplications available

    Practical demonstration:flow applications and cell sorting

    Workshop: Flow Cytometry

  • What is flow cytometry (FACS or FCM)?ComponentsLight scatter parametersFluorescence and MulticolourCell cycle analysisApoptosis and necrosis assay Cell proliferation assaySortingWorkshop: Flow Cytometry

  • What is flow cytometry?The analysis of single particles, often cells, within a heterogeneous suspension

    Whole blood, Cell cultures, Separated tissue, Isolated nuclei, Bacteria/yeast/parasites, Algae & planktonSignal from individual particles is collected for analysis as they pass through a laser in a stream of fluid.Data displayed as events on histograms/dot plotsWorkshop: Flow Cytometry

  • Workshop: Flow Cytometry

  • Components of a flow cytometerWorkshop: Flow Cytometry

  • Vital that cells pass through the laser bean in single suspension Cells injected into a flowing stream of saline solution (sheath fluid) Hydrodynamic focusing Compresses cell stream to approx 1 cell diameter Allows single cells to be interrogated by the laserOptimal imaging of cells is achieved with a low flow rate and high concentration of sampleFLUIDICSWorkshop: Flow Cytometry

  • Components of a flow cytometerWorkshop: Flow Cytometry

  • IntensityCounthLow signal height

    High signal heightWorkshop: Flow Cytometry

  • Size and granularity using flow cytometryWorkshop: Flow Cytometry

  • Cytometer Optical system comprises:Dichroics and FiltersWorkshop: Flow Cytometry

  • FITCFITCFITCFITCFITCFITCFITCFITCFITCEmitted fluorescence intensity is proportional to binding sitesFITC Log scale of Fluorescent IntensityNumber of EventsFluorescenceWorkshop: Flow Cytometry

  • FACS machines use lasers as sources for excitation; fixed single wavelength. Fluorescent light emission collected using filters as before. Therefore have to use flurophores compatible with lasers employed: FACSCalibur/FACSAria 488 and 647nm lasers.Workshop: Flow Cytometry

  • Emission is collected through emission filters positioned within the optical system of the flow cytometer.Workshop: Flow Cytometry

  • Dyes suitable for use on flow cytometers:

    488 excitation:FITC, Alexa 488, GFP, YFPPE, PI, RFP, PerCP, 7-AAD, PE-Cy5*, PE-Cy7*

    633nm excitation:APC, TOPRO-3, Cy5, Cy7

    * tandem dyes

  • Compensation FITC-Fluorescence Overlap

    650nm700nm500nm600nmRelative IntensityWavelength (nm)550nmWorkshop: Flow Cytometry

  • FITCPE650nm700nm500nm600nmRelative IntensityWavelength (nm)550nm24.8% of the FITC signal subtracted from PE.On a FacsCalibur flow cytometer, there is no provision to subtract FITC signal from PerCP.FITCPEPerformCompensationWorkshop: Flow Cytometry

  • Compensation PE-Fluorescence OverlapFITCPE650nm700nm500nm600nmRelative IntensityWavelength (nm)550nm750nm800nmPEPerCPWorkshop: Flow Cytometry

  • OptimalCompensationUnderCompensationOverCompensation16-colour compensation possible now on latest 3-laser, multi-parameter cytometersWorkshop: Flow Cytometry

  • Applying Gates for sub-population analysisSimple gating stratagiesWorkshop: Flow CytometryWhole blood light scatterGate on lymphocytes(light scatter)Assess T-cell population(fluorescence)

  • to more complex!

  • Applications of flow cytometry in research

    Multicolour analysisCell cycle Cell proliferationApoptosis and Cell ViabilityCell SortingMultiplex analysisWorkshop: Flow Cytometry

  • Applications of flow cytometry in research

    Multicolour analysisImmunophenotypingCells surface antigen detection (e.g. receptors, adhesion molecules)Intracellular stainingAssessing infection/transfection levelsAntibodies/ dyes/ Quantum dots

    Workshop: Flow Cytometry

  • Immunophenotypinge.g. diagnosis of leukaemiaCOMBINATION POPULATION IDENTIFIED CD4+/CDw29+ Helper/effector, more mature memory cells CD4+/CD45R+ Suppressor inducer, less mature non-memory cells CD4+/Leu8+ Suppressor inducer, some helper function CD4+/Class II MHC Activated cells, immature cells CD4+/CD25+ Activated cells (IL2 receptor) CD4+CD38+ Immature cells, activated cells CD8+/CD11b+ Of the CD11b+ cells the suppressors are bright CD8+ and NK are dim CD8+ CD8+/CD28+ Cytotoxic precursor/effector cells CD8+/CD57+ Cytotoxic function CD8+/Class II MHC+ Activated cells, immature cells CD8+/CD25+ Activated cells (IL2 receptor) CD8+/CD38+ Immature cells, activated cells CD16+/CD57+ Low NK activity CD16+/CD56+ Most potent NK activity Workshop: Flow Cytometry

  • Stem Cell Characterisation Clinical Application CD34+ Stem Cell Enumeration Method of repopulating stem cells following radiotherapy treatment Patient treated to produce excessive levels of pluripotent cells which are harvested from peripheral blood Number of cells reintroduced important in succsss rate of procedure Abs vs stem cell markers CD34 and CD45 used in enumeration procedure

  • Cell Cycle Analysis

  • Cell Cycle AnalysisDNA probes

    DAPI}Hoechst}UV

    Propidium iodide (PI)}7-AAD}488

    TOPRO-3}DRAQ5}633

    These dyes are stoichiometric number of bound molecules are equivalent to the number of DNA molecules presentThe cell cycleNote the cell volume (size) and DNA concentration change as the cell progresses through the cell cycleWorkshop: Flow Cytometry

  • A typical DNA histogramStoichiometric DNA probe bindingWorkshop: Flow Cytometry

  • Measuring height against width gives us area

    Two G1 cells together will have the same PI intensity as a G2 cell, but the area (signal h x w) will be greater and therefore can be discriminated on a plot of signal width vs areaWorkshop: Flow Cytometry

  • Cell Cycle Analysis:Bromodeoxyuridine (BrdU) incorporationA limitation to standard single colour DNA staining is that we cant determine whether S-phase cells are actually cycling

    Cells take up BrdU during S-phase, but not during G1 or G2, an Ab vs BrdU then allows us to determine which cells are actively cycling within a population by two-colour analysis:hLimitations. hInvitrogen Click-it EdU systemWorkshop: Flow Cytometry

  • Pulse-label with BrdU and taking samples at specific time points allows us to determine how cells behave kinetically through the cell cycle.Workshop: Flow CytometryAssessing cell proliferation: BrdU incorporation

  • Assessing cell proliferation using flow cytometryCFSE loaded cellsWorkshop: Flow Cytometry

  • Apoptosis and Cell Viability

  • Apoptosis Gene directed cell death An event that occurs during development and a response to trauma or disease Cancer cells develop a strategy to evade apoptosisApoptosis results in a number of cellular events that can be analysed by FACS:Fragmentation of DNA (subG1 assay, Hoechst dyes)Membrane structure and integrity Annexin-V, PI)Mitochondrial function (Mitotracker Red)Caspase activity (antibodies assay)Workshop: Flow Cytometry

  • Sub G1 apoptosis assaySub-G1 peakDNA fragmentation allows apoptosis to be quickly assessed with eg. PICan be seen as a population of small peaks to the left of G1 in a histogramQuick and easy way to determine if apoptosis is occurringWorkshop: Flow Cytometry

  • Workshop: Flow CytometryApoptosis detection using viability dye uptakeChanges in membrane permeability due to apoptosis allow intracellular dyes to stain unfixed cells

    7-AAD (DNA)

    Live cells exclude dye

    Apoptotic cells stain 7-AADdim

    Dead cells stain 7-AADbright

  • Annexin-V/PI assay for apoptosis:hPS normally on inside of cellular membrane hAnnV can bind to externalised PS highlighting cells that are apoptotic hPI will only go into cells with compromised membranes dead (necrotic) cellsWorkshop: Flow Cytometry

  • Membrane potential of the organelle reducedMitochondrial activity appears to change in parallel with cytoplasmic and plasma membrane eventsDyes that accumulate in mitochondria can therefore play role in detecting apoptosis -Mitotracker Red CMXRos-JC-1-DiOC2(3)-Laser Dye Styryl-751 (LDS-751) Reagent combinations can provide a window on intracellular processes not available with the much used pairing of annexin V and propidium iodide

    Apoptosis Organelle AnalysisWorkshop: Flow Cytometry

  • (CCCP) carbonyl cyanide m-chlorophenyl hydrazone

    Mitotracker Red can be loaded into live cells and taken up by mitochondriaLoss of membrane potential causes apoptotic cells to loose dye from organelleShift in fluorescence intensity indicates compromised mitochondriaWorkshop: Flow CytometryAlternative: DiOC6(3) for green fluorescent labelled mitochondria

  • Live/Dead assay

    Utilise the properties of dyes that are impermeable to intact cell membranes:Propidium iodideDAPITOPRO-3

    +ve fluorescence indicates compromised cell membranes and therefore dead cellsYeast cells + TOPRO-3Dead cells show more granularity and reduced sizeLive cells retain their morphology and appear larger in size and less granular Workshop: Flow Cytometry

  • Cell mediated cytotoxicity assay Dye exclusion assay to assess cell death, PKH26 (Sigma) Example: tumour cells (target) and NK cells (effector) Positive cytotoxic event recorded as an increase in cell fluorescence No requirement for radioisotopes e.g. 51Cr-release assay Also cell by cell assay - accurateSingle parameter histograms

  • Cell Sorting

  • Cell sortingAllows rare populations to be isolated from heterogenous populations (cell culture, blood samples, etc)Can isolate sub cellular particles (e.g. endosomes, nucleus, chromosomes)Allows transfection experiments to be enriched and single cell clones to be isolatedCan produce purity >95%

    Workshop: Flow Cytometry

  • Cell Sorting Transfected Cells Improve transefection efficiency siRNA knock down Stable cell line production Rare population isolation Single cell cloning Isolate spcific cell types from tissue preps Up to 4 populations simultaneously Various collection tubes and platesWorkshop: Flow Cytometry

  • Multiplex beadsWorkshop: Flow Cytometry

  • Workshop: Flow CytometryFluorescent capture bead technology Beads of various fluorescent intensities Can be conjugated with antibodies or biotin Multiplex conjugated kitsBender MedSysytemsBeckton DickinsonBeckman CoulterLuminexQiagenUpstate

    ELISA principals

  • Workshop: Flow Cytometry

  • Fluorescent proteins and their applications in bioimagingWorkshop: Flow Cytometry

  • What can we do with fluorescent proteins?Use as reporter genes to identify gene activationStudy transfection rates / successExpression of tagged proteins -Placed in-frame with gene of interestCompare expression / localisation against function (combine FACS with imaging)Environmental indicators (pH)Protein-protein interactions (FRET, split-GFP)Workshop: Flow Cytometry

  • Disadvantages of fluorescent proteins?SizeArtefactsMis-targettingOver expression Cell toxicitypH sensitiveAlways ensure adequate controlsN and C terminus constructsCheck functionality vs WT (if possible)Dont always select/gate brightest cells! Be objectiveStable cell lines? Transgenics?Alternative expression vectorWorkshop: Flow Cytometry

  • SummaryFlow cytometry is a powerful method for rapidly quantitating cellular fluorescenceA number of functional assays such as cell cycle and apoptosis can be determined by flow and can be used as a method for assessing e.g. the effects of drugs on cell function, or the expression of mutant proteinsFinally, cells and sub-cellular particles can be sorted from heterogeneous samples to yield near homogeneous populations for subsequent culturing or analysis.

    Workshop: Flow Cytometry

    Basics: light microscopy, fluorescence and epifluorescence, image resolution,Confocal microscopyDigital deconvolution microscopyDigital images basicsMulticolour experimental designFluorescent dyes for labelling cellular structuresFluorescent proteins in experiments