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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. Workshop: Flow Cytometry. LBFF: Flow Cytometry Facility Details. Location: Garstang level 8 Manager: Dr Gareth Howell

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LBFF: Leeds Bioimaging and Flow Cytometry Facility

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  1. Workshop: Flow Cytometry LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop – Flow Cytometry: Basic concepts, applications and experimental design

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Workshop: Flow Cytometry

  8. Electronics Fluidics Optics (detectors) Optics (lasers) Workshop: Flow Cytometry Components of a flow cytometer

  9. 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

  10. Electronics Workshop: Flow Cytometry Components of a flow cytometer

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

  12. Side scatter Forward scatter Workshop: Flow Cytometry Size and complexity using flow cytometry

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

  14. Workshop: Flow Cytometry Fluorescence 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

  15. 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

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

  17. 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

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

  19. FITC 530/30 PE 585/42 PerCP 670/LP Workshop: Flow Cytometry Perform Compensation PE PE FITC FITC 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, referred to as cross-beam compensation. 500nm 600nm 650nm 700nm 550nm Wavelength (nm)

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

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

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

  23. …to more complex!

  24. 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

  25. Immunophenotyping e.g. diagnosis of leukaemia Workshop: Flow Cytometry 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

  26. Stem Cell Characterisation

  27. 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/

  28. 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/

  29. 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

  30. Cell Cycle Analysis

  31. Workshop: Flow Cytometry • Cell Cycle Analysis 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

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

  33. H H x W = Area Intensity W Time Workshop: 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 area

  34. S-phase BrdU-FITC G2 G1 PI Workshop: Flow Cytometry Cell Cycle Analysis: Bromodeoxyuridine (BrdU) incorporation • 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

  35. 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.

  36. Apoptosis and Cell Viability

  37. 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)

  38. 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

  39. AnnV-FITC PS PI X X X X X X 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

  40. 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

  41. 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

  42. 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 Live cells retain their morphology and appear larger in size and less granular Dead cells show more granularity and reduced size

  43. 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

  44. Assessing cell proliferation

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

  46. 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

  47. 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%

  48. 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

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

  50. 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)

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