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Workshop: Flow Cytometry LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop – Flow Cytometry: Basic concepts, applications and experimental design Workshop: Flow Cytometry LBFF: Flow Cytometry Facility Details Location: Garstang level 8 Manager: Dr Gareth Howell http://www.fbs.leeds.ac.uk/facilities/ flowcytometry/ E: [email protected] T: x37270 My Office Workshop: Flow Cytometry BD FACSCalibur 2-laser, 4 colour analyser cytometer Fixed emission filter set-up BD FACSAria 2-laser, 7 colour analyser and cells sorting cytometer Interchangable emission filter set-up Partec PASIII Single laser, 4 colour analyser cytometer HBO (mercury) lamp Interchangable filter set-up Workshop: Flow Cytometry Purpose of this workshop: To introduce the concepts of flow cytometry (FACS)analysis To illustrate the role FACS can play in your research Demonstrate the capabilities of FACS Experimental design To discuss the limitations of FACS Seminar: Introduction to FACS Applications available Practical demonstration: FACS applications and cell sorting Workshop: Flow Cytometry • • • • • • • • What is flow cytometry? Components Size and complexity using flow cytometry Fluorescence and Multicolour flow cytometry Cell cycle analysis Apoptosis and necrosis assay Cell proliferation assay Sorting Workshop: 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 & plankton • Signal 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 plots Workshop: Flow Cytometry Workshop: Flow Cytometry Components of a flow cytometer Electronics Fluidics Optics (lasers) Optics (detectors) Workshop: Flow Cytometry FLUIDICS • 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 laser •Optimal ‘imaging’ of cells is achieved with a ‘low’ flow rate and high concentration of sample Workshop: Flow Cytometry Components of a flow cytometer Electronics Laser Voltage Workshop: Flow Cytometry Low signal height High signal height Laser Voltage Time Laser Count Voltage Time h Time Intensity Workshop: Flow Cytometry Size and complexity using flow cytometry Side scatter Forward scatter Workshop: Flow Cytometry Cytometer Optical system comprises: Dichroics and Filters Fluidics Detectors Workshop: Flow Cytometry Fluorescence Emitted fluorescence intensity is proportional to binding sites FITC FITC FITC FITC Number of Events FITC FITC FITC FITC Log scale of Fluorescent Intensity FITC FITC Workshop: 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. APC Workshop: Flow Cytometry Emission is collected through emission filters positioned within the optical system of the flow cytometer. APC Dyes suitable for use on flow cytometers: • 488 excitation: – FITC, Alexa 488, GFP, YFP – PE, PI, RFP, – PerCP, 7-AAD, PE-Cy5, PE-Cy7 • 633nm excitation: – APC, TOPRO-3, Cy5, Cy7 Workshop: Flow Cytometry Compensation FITC-Fluorescence Overlap PE 585/42 PerCP 670/LP Relative Intensity PE FITC 530/30 PerCP FITC 500nm 550nm 600nm 650nm Wavelength (nm) 700nm FITC Workshop: Flow Cytometry Perform Compensation FITC PE 585/42 PerCP 670/LP Relative Intensity FITC 530/30 FITC 24.8% of the FITC signal subtracted from PE. On a FacsCalibur flow cytometer, there is no provision to subtract FITC signal from PerCP, referred to as cross-beam compensation. 500nm 550nm 600nm 650nm Wavelength (nm) 700nm Workshop: Flow Cytometry Compensation PE-Fluorescence Overlap PerCP 670/LP Relative Intensity PE FITC PE 530/30 585/42 PerCP FITC 500nm 550nm 600nm 650nm Wavelength (nm) 700nm 750nm 800nm PE Workshop: Flow Cytometry Optimal Compensation Under Compensation Over Compensation 16-colour compensation possible now on latest 3-laser, multi-parameter cytometers Workshop: Flow Cytometry Applying Gates for sub-population analysis Simple gating stratagies… Whole blood light scatter Gate on lymphocytes (light scatter) Assess T-cell population (fluorescence) …to more complex! Workshop: Flow Cytometry Applications of flow cytometry in research • • • • • • Immunophenotyping Stem cell characterisation Cell cycle Apoptosis and Cell Viability Cell proliferation (CFSE, BrdU/Hoechst) Cell Sorting Workshop: Flow Cytometry • Immunophenotyping e.g. diagnosis of leukaemia COMBINATION 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 Stem Cell Characterisation • Stem Cell Characterisation Functional analysis • • • • Cytosolic aldehyde dehydrogenase (ALDH) activity High levels found in stem cells Drug resistance Cleavable enzyme assay (AldeFluor, StemCell Tech.) http://science.cancerresearchuk.org/ • Stem Cell Characterisation Side population analysis • Efficient membrane pumps • Exclude dyes e.g. Rhodamin 123 and Hoechst dye • Hoechst dyes bind DNA in live cells (blue and red fluorescence) • UV excitation • Pumped out by ABC (ATPase Binding Cassette) • Stem cells can be characterised by low side populations –ve for Hoechst dye. • Membrane markers to confirm. http://science.cancerresearchuk.org/ • 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 Workshop: Flow Cytometry •Cell Cycle Analysis DNA probes DAPI Hoechst The cell cycle } } 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 present Note the cell volume (size) and DNA concentration change as the cell progresses through the cell cycle Workshop: Flow Cytometry Stoichiometric DNA probe binding l A typical DNA histogram Workshop: Flow Cytometry H H x W = Area W Time 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 area Workshop: Flow Cytometry •A limitation to standard single colour DNA staining is that we can’t determine whether Sphase cells are actually cycling BrdU-FITC Cell Cycle Analysis: Bromodeoxyuridine (BrdU) incorporation S-phase G2 G1 •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 system PI Workshop: 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. Apoptosis and Cell Viability Workshop: Flow Cytometry •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 apoptosis Apoptosis 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 • Quick and easy apoptosis assay: Sub-G1 DNA fragmentation allows apoptosis to be quickly assessed with eg. PI Can be seen as a population of small peaks to the left of G1 in a histogram Quick and easy way to determine if apoptosis is occurring Sub-G1 peak Workshop: Flow Cytometry 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) cells AnnV-FITC PS PI Workshop: Flow Cytometry •Apoptosis – Organelle Analysis •Membrane potential of the organelle reduced •Mitochondrial activity appears to change in parallel with cytoplasmic and plasma membrane events •Dyes 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 muchused pairing of annexin V and propidium iodide Workshop: Flow Cytometry •Mitotracker Red can be loaded into live cells and taken up by mitochondria •Loss of membrane potential causes apoptoic cells to loose dye from organelle •Shift in fluorescence intensity indicates compromised mitochondria (CCCP) carbonyl cyanide m-chlorophenyl hydrazone Alternative: DiOC6(3) for green fluorescent labelled mitochondria Workshop: Flow Cytometry Yeast cells + TOPRO-3 Live/Dead assay Utilise the properties of dyes that are impermeable to intact cell membranes: Propidium iodide DAPI TOPRO-3 +ve fluorescence indicates compromised cell membranes and therefore dead cells Dead cells show more granularity and reduced size Live cells retain their morphology and appear larger in size and less granular • 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 accurate Single parameter histograms Assessing cell proliferation Workshop: Flow Cytometry Assessing cell proliferation using flow cytometry CFSE loaded cells Assessing cell proliferation using flow cytometry BrdU/Hoechst quenching assay DNA binding dye Hoechst fluorescence quenched if BrdU incorporated into DNA Can be used to assess cell proliferation PI not quenched – allows determination of cell cycle as before. Requires flow cytometer with UV excitation Needs careful optimization of BrdU labelling Diermeier et al (2004) Cell Prolif. 37:195 Workshop: Flow Cytometry • Cell sorting – Allows 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 isolated – Can produce purity >95% Cell Sorting Chromosomes • Chromosome specific DNA libraries, DNA for sequencing, probes for reverse painting, array painting. • Many lymphomas have chromosomal abnormalities. • Base specific dyes allow chromosomes to be separated on dot plots http://www.chrombios.com/Service/ServiceFACS.html Workshop: Flow Cytometry Fluorescent proteins and their applications in bioimaging Workshop: Flow Cytometry What can we do with fluorescent proteins? •Use as reporter genes to identify gene activation •Study transfection rates / success •Expression of tagged proteins -Placed in-frame with gene of interest •Compare expression / localisation against function (combine FACS with imaging) •Environmental indicators (pH) •Protein-protein interactions (FRET, split-GFP) Workshop: Flow Cytometry Disadvantages of fluorescent proteins? •Size •Always ensure adequate controls •Artefacts •N and C terminus constructs •Mis-targetting •Check functionality vs WT (if possible) •Over expression •Cell toxicity •pH sensitive •Don’t always select/gate brightest cells! Be objective •Stable cell lines? Transgenics? •Alternative expression vector Workshop: Flow Cytometry • Summary – Flow cytometry is a powerful method for rapidly quantitating cellular fluorescence – A 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 proteins – Finally, cells and sub-cellular particles can be sorted from heterogeneous samples to yield near homogeneous populations for subsequent culturing or analysis. Workshop: Flow Cytometry Wavelengths of visible light The wavelength of visible light ranges from 380 nm (violet) to 780 nm (red). Visible light spectrum UV IR Workshop: Flow Cytometry Fluorescence - basics Excitation Emission Fluorescence intensity Fluorescent molecules are characterised by their ability to absorb short wavelength light and emit at a longer wavelength. Wavelength (nm) Workshop: Flow Cytometry Visible light spectrum UV IR Bandpass eg 530/30 Bandpass eg 585/40 Longpass eg LP670 FITC, Alexa 488 Phycoerythrin (PE) Cy5, APC, PerCP Workshop: Flow Cytometry Emission Spectra 100% PE APC PerCP Normalized Intensity FITC 0% 400 500 600 Wavelength (nm) 700 800 Workshop: Flow Cytometry More Emission Spectra! Cascade Blue FITC Alexa 430 PE PI APC PerCP PerCP-Cy5.5 PE-Cy7 Normalized Intensity 100% 0% 400 500 600 Wavelength (nm) 700 800 Workshop: Flow Cytometry Designing Multicolour Experiments Why? •Allows a number of different structures (proteins / lipids / compartments) to be visualized at the same time • Can provide clues / evidence to the function of your protein of interest • Design principles can be applied to any fluorescent molecule: fluorescent protein, membrane marker, antibody or dye in live or fixed cells How? • Simply by studying the configuration of the imaging system and the excitation / emission characteristics of the proposed dyes one can design a multicolour fluorescent experiment Workshop: Flow Cytometry Red Graphic representations of fluorescence Green -ve/ Green +ve/ data Red +ve Red +ve Green Green -ve/ Red -ve Green +ve/ Red -ve