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Introduction To Flow Cytometry: Niga Nawroly [email protected] What is flow cytometry? Flow cytometry is a method of measuring multiple physical and chemical characteristics of particles by optical means. • Peripheral blood, Bone marrow cells • Bacteria • Yeast Applications of Flow Cytometry. • Cell size. • Cytoplasmic granularity. • Cell surface antigens (phenotyping). • Apoptosis. • Intracellular cytokine production. • Intracellular signalling. • Gene reporter (GFP). • Cell cycle, DNA content, composition, synthesis. • Bound and free calcium. • Cell proliferation (BRDU and CFSE) • Cell sorting, single cell cloning (clonecyt) Phenotyping, Size and granularity detection: Sample requirements: A suspension of single cells or other particles in a suitable buffer, usually PBS. Typical density : 105 - 107 cells / ml + Incubate Acquire A cytometer consists of: Electronics Fluidics Light detectors FL3 PerCP, Cy5 FL2 PE FL1 FITC SSC Laser Light 488nm FSC Computer Size and Granularity Size All Data shown is from: BAL from Balb/c primed with TT Data produced by Dr John Tregoning Presenting and Interpreting Data Single parameters can be displayed as a histogram. Dual parameter data can be displayed in two dimensions using dot, density or contour plots. Gating: Back-gating: Available analysis software • Cellquest • WinList • Flowjo • WinMDI Free cytometry software: http://flowcyt.cyto.purdue.edu/flowcyt/software/Catalog.htm Flow Cytometry : Reference Material Books • Practical flow Cytometry Howard M Shapiro. • Flow Cytometry: A Practical Approach MG Ormerod. • Introduction to Flow Cytometry, JV Watson. • Flow Cytometry: First Principals, Alice Givan. • Cytometric Analysis of Cell Phenotype and Function, McCarthy & Macey. Journal • Cytometry : Journal of the International Society for Analytical Cytology. www.cytometry.org Flow Cytometry on the web • ISAC (International Society For Analytical Cytology) www.isac-net.org • Salk Institute http://flowcyt.salk.edu • Purdue University www.cyto.purdue.edu • Scripps Research Institute http://facs.scripps.edu/index.html • Cancer Research UK http://science.cancerresearchuk.org/sci/facs Our website http://www.wfi.med.ic.ac.uk/Flow.htm New Website address: http://www1.imperial.ac.uk/medicine/about/institutes/wfi