Introduction To Flow Cytometry:

Niga Nawroly

n.nawroly@imperial.ac.uk

Flow cytometry is a method of measuring multiple physical and chemical characteristics of particles by optical means.

What is flow cytometry?

• Peripheral blood, Bone marrow cells• Bacteria• Yeast

• 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)

Applications of Flow Cytometry.

A suspension of single cells or other particlesin a suitable buffer, usually PBS.

Typical density : 105 - 107 cells / ml

+

Incubate Acquire

Phenotyping, Size and granularity detection:

Sample requirements:

LaserLight488nm

FL2 PE

SSC

FSC

Fluidics

Electronics

Computer

FL3 PerCP, Cy5

FL1 FITC

A cytometer consists of:

Light detectors

Size and Granularity

Size

Gra

nu

lari

ty

All Data shown is from:BAL from Balb/c primed with TTData produced by Dr John Tregoning

Presenting and Interpreting Data

Dual parameter data can be displayed in two dimensions using dot, density or contour plots.

Single parameters can be displayed as a histogram.

Gating:

Back-gating:

• Cellquest• WinList • Flowjo• WinMDI

http://flowcyt.cyto.purdue.edu/flowcyt/software/Catalog.htm

Available analysis software

Free cytometry software:

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

• 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

Flow Cytometry on the web

http://www.wfi.med.ic.ac.uk/Flow.htm

New Website address:

http://www1.imperial.ac.uk/medicine/about/institutes/wfi

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