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
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