Introduction To Flow Cytometry:

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Presentation transcript:

Introduction To Flow Cytometry: Niga Nawroly n.nawroly@imperial.ac.uk

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 Computer FL2 PE FL1 FITC SSC Laser Light 488nm FSC

Size and Granularity 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 New Website address: http://www.wfi.med.ic.ac.uk/Flow.htm http://www1.imperial.ac.uk/medicine/about/institutes/wfi