Presentation on theme: "What is Flow Cytometry? Introduction to Flow Cytometry"— Presentation transcript:
1 What is Flow Cytometry? Introduction to Flow Cytometry IGC WorkshopApplications in Flow CytometryRui GardnerIGC – April 29, 2010
2 Outline Potential Applications of Flow Cytometry Cell State Cell FunctionImmunophenotypingCell activationCell cycleCell proliferationApoptosisDifferentiationIdentification of “stem cells”Cytokine SecretionActivation of signalling pathwaysCalcium fluxLevels of intracellular reactive oxygen speciesTelomere lengthVantagens da Citometria de fluxoEstatistica populacionalcell sortingAnalise de populaçoes rarasAplicaçoesCell SeparationSorting
11 Propidium Iodide plus BrdU staining Cell Cycle - Bromodeoxyuridine (BrdU) methodPropidium Iodide plus BrdU staining• Thymidine analog• Taken up by cells in S-phase• Usually in combination with Propidium iodide104S Phase103Anti-BrdU FITC102101G1G2/MPropidium IodideNew Click-It DNA technology from Invitrogen does not require DNA denaturation.
12 Pyronin Y plus Hoechst 33342/33258 Cell Cycle - G0/G1 discriminationPyronin Y plus Hoechst 33342/33258G0/G1SG2/MCell CountG0SG2/MG1RNA ContentLive Go cell cycle analysis with Hoechst and Pyronin YFlow cytometry may be used to determine the level of senescence in a cell population. Go and G1 can be separated by use of Pyronin Y (Sigma) which preferentially binds to RNA and Hoechst (Sigma) which binds to A-T base pairs. Cells are loaded with Hoechst at 10 ug/ml and Pyronin Y at 0.5 ug/ml by incubating for 45 mins at 37C. Pyronin Y is excited at 488 nm and emits at 575 nm, while Hoechst is excited UV lasers ( nm), near UV line (375 nm) or violet diode (405 nm).
22 Tracking Cell Proliferation with CFSE Dilution of CFSECell DivisionsSTAIN WITH CFSECELLCFSE: Carboxy-fluorescein diacetate, succinimidyl ester (fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties)Diffuses freely into cellsIntracellular esterases cleave the acetate groups converting CFSE into a fluorescent, membrane impermeant dye.Not transferred to adjacent cells. Retained by the cell in the cytoplasmDoes not adversely affect cellular functionDuring each round of cell division, the relative intensity of the dye is decreased by half.
31 Each type of bead coated with capture antibody for particular cytokine Multiplex Bead Arraysbead coated with capture antibody for particular cytokineCytokinesCan use arrays of beads to measure multiple cytokines at once on the one sampleEach type of bead coated with capture antibody for particular cytokineMix beads with sample, wash, then add anti-cytokine ab with fluorchrome reporterAmount of fluorescence measured proportional to amount of cytokine bound to each bead (equivalent to ELISA OD reading)Amount Cytokine
34 Calcium FluxFluorescence-imaging of human erythrocytes treated with PGE2 using the calcium fluorophor Fluo-4Effects of T cell receptor stimulation on CD4 cell ionized calcium concentration ([Ca2+]i).
43 Establishing Fluorescent Cell Lines Carina Santos (IMM)InterphaseCulturedmCherry signalSortedAnaphaseHuman hepatoma cell lineExpressing α-tubulin fused with mCherrymCherry signalmCherry signal
44 Chromosome sorting Human cell line with translocation between chromosome 2 and chromosome 17Normal human cell lineAT-rich DNA signalGC-rich DNA signal
45 Establishment of Cell Clones Sort single cell into each welltimeClone AClone BClone C
46 Future AdvancesMore colours for immunofluorescence (quantum dots, tandem dyes)Reduced laser size and capillary flow techniques mean smaller instrumentsInstruments can now image cell at point of laser interrogation