Flow Cytometry Workshop Insert Date Dr Gareth Howell g.j.howell@leeds.ac.uk x37270

LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop: Flow Cytometry LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop – Flow Cytometry: Basic concepts, applications and experimental design Basics: light microscopy, fluorescence and epifluorescence, image resolution, Confocal microscopy Digital deconvolution microscopy Digital images basics Multicolour experimental design Fluorescent dyes for labelling cellular structures Fluorescent proteins in experiments

LBFF: Flow Cytometry Facility Details 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: g.j.howell@leeds.ac.uk T: x37270 My Office

Workshop: Flow Cytometry BD FACSAria 2-laser, 7 colour analyser and cells sorting cytometer Interchangable emission filter set-up BD FACSCalibur 2-laser, 4 colour analyser cytometer Fixed emission filter set-up Partec PASIII Single laser, 4 colour analyser cytometer HBO (mercury) lamp Interchangable filter set-up

Purpose of this workshop: Workshop: Flow Cytometry Purpose of this workshop: To introduce the concepts of flow cytometry (FACS)analysis To illustrate the role flow can play in your research Demonstrate the capabilities of flow Experimental design To discuss the limitations of flow Seminar: Introduction to flow Applications available Practical demonstration: flow applications and cell sorting

What is flow cytometry (FACS or FCM)? Components Workshop: Flow Cytometry What is flow cytometry (FACS or FCM)? Components Light scatter parameters Fluorescence and Multicolour 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 Electronics Components of a flow cytometer Fluidics Optics (detectors) (lasers)

FLUIDICS Workshop: 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 laser Optimal ‘imaging’ of cells is achieved with a ‘low’ flow rate and high concentration of sample

Workshop: Flow Cytometry Electronics Components of a flow cytometer

Workshop: Flow Cytometry Laser Time Voltage Low signal height High signal height Count h Intensity

Size and granularity using flow cytometry Workshop: Flow Cytometry Size and granularity using flow cytometry Forward scatter Side scatter

Workshop: Flow Cytometry Cytometer Optical system comprises: Dichroics and Filters Fluidics Detectors

Fluorescence Workshop: Flow Cytometry Emitted fluorescence intensity is proportional to binding sites FITC FITC FITC FITC FITC FITC FITC FITC FITC FITC Number of Events Log scale of Fluorescent Intensity

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.

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

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 * tandem dyes

Compensation FITC-Fluorescence Overlap Workshop: Flow Cytometry Compensation FITC-Fluorescence Overlap FITC 530/30 PE 585/42 PerCP 670/LP FITC PE PerCP Relative Intensity 500nm 550nm 600nm 650nm 700nm Wavelength (nm)

Workshop: Flow Cytometry Perform Compensation PE PE FITC FITC FITC 530/30 PE 585/42 PerCP 670/LP Relative Intensity 24.8% of the FITC signal subtracted from PE. On a FacsCalibur flow cytometer, there is no provision to subtract FITC signal from PerCP. 500nm 550nm 600nm 650nm 700nm Wavelength (nm)

Compensation PE-Fluorescence Overlap Workshop: Flow Cytometry Compensation PE-Fluorescence Overlap FITC 530/30 PE 585/42 PerCP 670/LP PE FITC Relative Intensity PerCP 500nm 550nm 600nm 650nm 700nm 750nm 800nm Wavelength (nm) PE

Optimal Compensation Under Compensation Over Compensation Workshop: Flow Cytometry Optimal Compensation Under Compensation Over Compensation 16-colour compensation possible now on latest 3-laser, multi-parameter cytometers

Assess T-cell population Workshop: Flow Cytometry Applying Gates for sub-population analysis Simple gating stratagies… Gate on lymphocytes (light scatter) Assess T-cell population (fluorescence) Whole blood light scatter

…to more complex!

Applications of flow cytometry in research Workshop: Flow Cytometry Applications of flow cytometry in research Multicolour analysis Cell cycle Cell proliferation Apoptosis and Cell Viability Cell Sorting Multiplex analysis

Applications of flow cytometry in research Workshop: Flow Cytometry Applications of flow cytometry in research Multicolour analysis Immunophenotyping Cells surface antigen detection (e.g. receptors, adhesion molecules) Intracellular staining Assessing infection/transfection levels Antibodies/ dyes/ Quantum dots

e.g. diagnosis of leukaemia 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 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 Analysis Workshop: Flow Cytometry DNA 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 present The cell cycle Note the cell volume (size) and DNA concentration change as the cell progresses through the cell cycle

Workshop: Flow Cytometry Stoichiometric DNA probe binding A typical DNA histogram l

Workshop: Flow Cytometry Time Intensity H H x W = Area W 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

Bromodeoxyuridine (BrdU) incorporation Workshop: Flow Cytometry Cell Cycle Analysis: Bromodeoxyuridine (BrdU) incorporation PI BrdU-FITC S-phase G1 G2 A limitation to standard single colour DNA staining is that we can’t 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 system

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

Assessing cell proliferation using flow cytometry Workshop: Flow Cytometry Assessing cell proliferation using flow cytometry CFSE loaded cells

Apoptosis and Cell Viability

Apoptosis Workshop: Flow Cytometry 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)

Sub G1 apoptosis assay Workshop: Flow Cytometry Sub-G1 peak 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

Apoptosis detection using viability dye uptake Workshop: Flow Cytometry Apoptosis detection using viability dye uptake Changes 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

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 X PI

Apoptosis – Organelle Analysis 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 much used 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 apoptotic 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

Live/Dead assay Workshop: Flow Cytometry Yeast cells + TOPRO-3 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 Live cells retain their morphology and appear larger in size and less granular Dead cells show more granularity and reduced size

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

Cell Sorting

Cell sorting Workshop: Flow Cytometry 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 Transfected Cells 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 plates

Workshop: Flow Cytometry Multiplex beads

Fluorescent capture bead technology Workshop: Flow Cytometry Fluorescent capture bead technology Beads of various fluorescent intensities Can be conjugated with antibodies or biotin Multiplex conjugated kits Bender MedSysytems Beckton Dickinson Beckman Coulter Luminex Qiagen Upstate ELISA principals

Workshop: Flow Cytometry Coated latex bead (FL1) FL1 FL2 Analyse by flow cytometry using bivariant dot plot Incubate with e.g. cell lysate Y Incubate with FL2 labelled antibody vs protein of interest

Fluorescent proteins and their applications in bioimaging Workshop: Flow Cytometry Fluorescent proteins and their applications in bioimaging

What can we do with fluorescent proteins? 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)

Disadvantages of fluorescent proteins? Workshop: Flow Cytometry Disadvantages of fluorescent proteins? Size Artefacts Mis-targetting Over expression Cell toxicity pH sensitive Always ensure adequate controls N and C terminus constructs Check functionality vs WT (if possible) 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.