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UNIVERSITY OF CALGARY FLOW CYTOMETRY FACILITY

UNIVERSITY OF CALGARY FLOW CYTOMETRY FACILITY. ‘Efficient and reliable flow cytometry services with the highest standards of quality and productivity’.

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UNIVERSITY OF CALGARY FLOW CYTOMETRY FACILITY

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  1. UNIVERSITY OF CALGARY FLOW CYTOMETRY FACILITY ‘Efficient and reliable flow cytometry services with the highest standards of quality and productivity’

  2. Flow cytometry is a technique for measuring physical and chemical properties of individual cells as they travel in suspension one by one past a sensing point.

  3. BASICS OF FLOWCYTOMETRY • The cells in suspension are forced to pass in a fluid stream through a flow cell. • The fluid stream intersects the focus of a laser. • The laser light is scattered and, if the cells are fluorescent, they produce fluorescent signals. • These light signals are then converted to electronic signals (voltages).

  4. INSTRUMENTATION • Fluidics • Optics • 3. Electronics

  5. FLUIDICS • The purpose of the fluidics system is to transport cells in a fluid stream to the laser beam for interogation.

  6. OPTICS The optics system consist of a laser to illuminate the cells in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors (I.e . to resolve different colors).

  7. The flow of sheath fluid restricts the cells to the center of the sample core for optimal illumination (hydrodynamic focusing). • Only one cell should move through the laser beam at a given moment.

  8. “THE LASER” • Lasers emit coherent light, in a fine, straight beam at a specified wavelength. • The use of a laser allows the beam of light to be focused on single cells so that basic measurements based on beam disturbance can be taken (FSC, SSC) . • LASER: Acronym for • Light Amplification by • Stimulated Emission of • Radiation

  9. ELECTRONICS • Processing of signals from detectors -Cells passing through the laser beam generate light signals.These light signals are then converted to electronic signals (voltages). -The electrical voltage generated will be proportional to the number of photons (amount of light) emitted by the cell/particle. -The voltages are processed by the computer.

  10. The sample is injected into a stream of sheath fluid within the flow chamber; the sample core remains separate but coaxial within the sheath fluid.

  11. Light is bent (diffracted) depending on the size and refractive index of the cell • Detected along the axis of incident light • (0-100)

  12. Light is reflected/bounced to the side • Proportional to cell granularity • Detected at 90o to incident light axis

  13. When cells pass through the laser intercept, they scatter laser light andthe system measures the degree and direction of scattered light-indicators of the cell’s size, shape and structure.

  14. What do the scatter signals tell us? • Together the forward and side scatter signals can provide useful ways to characterize different cell types. • Example:Leucocytes (white blood cells).

  15. FLUOROSCENCE • Fluorescent compounds absorb light energy over a range of wavelengths, which causes an electron in the compound to be raised to a higher energy level. • The excited electron emits this excess energy as a photon of light. • While each fluorochrome will have an optimal, or peak emission wavelength, the spectra will actually be distributed over a number of wavelengths.

  16. Any fluorescent molecules present on the cell fluoresce.

  17. Injector Tip Sheath fluid Fluorescence signals Focused laser beam • The cells are forced to pass in a fluid stream through a flow cell where the fluid stream intersects the focus of the laser.

  18. More than one fluorochrome can be used simultaneously if each is excited at 488 nm and if the peak emission wavelengths are not extremely close to each other.

  19. DATA DISPLAY Negative Control Sample Histogram: Relative fluorescence vs. # of events

  20. DATA DISPLAY Quadrants can be applied to any 2 parameter display.

  21. IMMUNOPHENOTYPING Antibody-fluorescent dye conjugates bind to antigens and the quantity of the fluorescent light emitted is correlated with the cellular marker in question.

  22. DATA DISPLAY

  23. Activation: Surface Receptor Expression Up Regulation of IL-2 Receptor on Mouse B Cells

  24. --- Isotype __ Anti-P-Selectin CELL ADHESION MOLECULES A. Unstimulated B. Thrombin-activated Expression of P-selectin is up-regulated on activated peripheral blood platelets.

  25. Activation: Intracellular Cytokines

  26. INTRACELLULAR CYTOKINE MEASUREMENT

  27. INTRACELLULAR CYTOKINE MEASUREMENT Multi-parameter flow cytometric analysis of cultured Th1 and Th2 cells

  28. APOPTOSIS

  29. METHODS FOR DETECTING APOPTOSIS: Annexin V assay

  30. Apoptosis: Annexin V Assay Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody

  31. PI vs. ANNEXIN • One of the more common uses of DNA-based dyes is to identify apoptotic cells. • Necrotic cells are widely permeable to a number of cell labels (usually PI) whereas apoptotic cells are impermeable. • Staining for apoptotic markers (i.e., Annexin V) will identify apoptotic cells, whereas necrotic cells will also stain with PI.

  32. PI vs. ANNEXIN

  33. Apoptosis: Quantitative Analysis of Caspase-3 Activation Jurkat cells Treated with Campothecin Caspase 3 PE

  34. Apoptosis: TUNEL Assay Jurkat Cells Treated for 6 hours with IgM Anti Fas Antibody

  35. Apoptosis: bcl-2 Regulation Mitochondrial Protein bcl-2 Blocks Apoptosis Bcl-2 Is Down Regulated During Apoptosis

  36. METHODS FOR DETECTING APOPTOSIS: Gene regulation Analysis of p53 expression in SV40 transformed rat ovarian cells

  37. METHODS FOR DETECTING APOPTOSIS: Histone Phosphorylation

  38. Proliferation: Nucleotide Analogs Bromodeoxyuridine Is Incorporated Into Cellular DNA By Pulsing Proliferating Cells The Nucleotide May Be Conjugated With Fluorochrome Or Detected By Antibodies

  39. PROLIFERATION: CFSE Carboxyfluorescein succinimidyl ester The amount of CFSE in the cell in the membrane of proliferating cells halves with each successive division and therefore, the fluorescence can be used to monitor the number of cell divisions.

  40. Ploidy: Nucleic Acid Dyes DAPI is one of many non-vital dyes that binds DNA on an equimolar basis This allows the precise quantitation of a cell populations proliferative index

  41. DNA & CELL CYCLE ANALYSIS PI: Key feature of DNA probes is that they are STOICHIOMETRIC. Total nuclear content and the fraction of cells in each phase of the cell cycle can be measured.

  42. Normal Cell Cycle G0 G1 G2 M 4N 2N M G0 G2 DNA Analysis G1 s Count s 0 200 400 600 800 1000 DNA content

  43. INTRACELLULAR CYTOKINE MEASUREMENT • The production of cytokines by specific cell types can be determined as opposed to measuring the amount of secreted cytokine present in the serum or supernatant.

  44. APOPTOSIS: SUBGENOMIC DNA PEAKS

  45. TRANSFECTION EFFICIENCY Using Green Fluorescent Protein (GFP) as a co-transformation marker is one of the most common applications of GFP-expressing vectors

  46. TRANSGENIC CELL LINE CELL NUMBER CONTROL 0 100 200 300 400 500 600 700 FLUORESCENCE

  47. ESTIMATING CELL VIABILITY PROPIDIUM IODIDE (PI) -Excluded by viable cells and when taken up by dying cells, binds to nucleic acids and fluoresces orange.

  48. Viability: Fluorescein Diacetate & PI FDA converted to fluorescent compound in live cells

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