Flowcytometry

1. Flowcytometry

2. What Is Flow Cytometry? • Flow ~ cells in motion • Cyto ~ cell • Metry ~ measure • Measuring properties of cells while in a fluid stream

4. Applications of Flow Cytometry. • Cell size. • Cytoplasmic granularity. • Cell surface antigens (Immunophenotyping). • Apoptosis. • Intracellular cytokine production. • Intracellular signalling. • Gene reporter (GFP). • Cell cycle, DNA content, composition, synthesis. • Bound and free calcium. • Cell proliferation • Cell sorting, single cell cloning

5. Principle of Flow Cytometry • Cells in suspension • Cells flow in single-file • Intercepted by light source(s) (laser) • Scatter light and emit fluorescence • Signal collected, filtered and • Converted to digital values • Storage on a computer Fluidics Optics Electronics Data display and analysis

6. Basic Principles of Flow Cytometry • Single cell or particle suspension • Fluorescent dyes or Abs that can be attached to an antigen or protein of interest • Flow cell, sheath fluid and a focused laser beam

7. SampleStream Cell The Flow Cell The introduction of a large volume into a small volume in such a way that it becomes “focused” along an axis is called Hydrodynamic Focusing. Sheath

8. Basic Principles cont’d • Light is either scattered or absorbed when it strikes a cell • Light scatter is dependent on the internal structure, size and shape. • Forward scatter = size of the cell • Side Scatter = complexity of the cell

9. Laser Beam FSCDetector Forward Scatter

10. Laser Beam FSCDetector Collection Lens SSC Detector Side Scatter

11. Side scatter Monocytes Lymphocytes Granulocytes Forward scatter

12. Granulocytes Lymphocytes SSC Monocytes RBCs, Debris, Dead Cells FSC Why Look at FSC v. SSC • Since FSC ~ size and SSC ~ internal structure, a correlated measurement between them can allow for differentiation of cell types in a heterogeneous cell population

13. Side scatter Monocytes Lymphocytes Granulocytes Forward scatter

14. FLOW CYTOMETRY - Cells are labeled withfluorescent antibodies directed against cell surface molecules - Usingdifferentcolorfluorochromes allows counting of many markers simultaneously and allows identification of several markers on the same cell ( Multiparameter Flow) - In the instrument, cells passone-by-onepast a laser to excite the fluorochromes and there are detectors for each type of fluorochrome

15. Flouresent tag Surface of a cell, e.g., a lymphocyte (in solution) - cells are labeled with fluoresence antibodies

16. What Happens in a Flow Cytometer

17. Fluorochrome

18. Reflected Dichroic Filter Passed Short or Long Pass Filter Band Pass Filter Adsorbed Absorption Filter Basic Principles cont’d • Fluorescent dyes absorb light of a specific wavelength and reemit light of a different wavelength • Fluorescent signals are detected by PMT and amplified • Optical filters are used to steer light of specific wavelengths to the photo detector

19. Detectors There are two main types of photo detectors used in flow cytometry Photodiodes Used for strong signals, when saturation is a potential problem (eg. FSC detector) Photomultiplier tubes (PMT) More sensitive than a Photodiode, a PMT is used for detecting small amounts of fluorescence emitted from fluorochromes.

20. Electronics • Electrical pulses are digitized, the data is stored (‘list mode data’), analysed and displayed through a computer system. • The end result is quantitative information about every cell analysed • Large numbers of cells can be processed quickly

21. CD19+ CD19+ CD3 - CD3 + CD19- CD19- CD3- CD3+ CD3- CD3+ CD19 CD19 CD3 CD3 THE OPERATOR SETS “GATES” DEFINING POSITIVE AND NEGATIVE FOR EACH MARKER. Therefore, you can define each cell counted with regard to CD19 or CD3 positivity. Note that there are not normally cells in the circulation that express both T and B cell surface markers.