introduction to flow cytometry: niga nawroly [email protected]
TRANSCRIPT
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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
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• 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.
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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:
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LaserLight488nm
FL2 PE
SSC
FSC
Fluidics
Electronics
Computer
FL3 PerCP, Cy5
FL1 FITC
A cytometer consists of:
Light detectors
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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
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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.
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Gating:
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Back-gating:
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• Cellquest• WinList • Flowjo• WinMDI
http://flowcyt.cyto.purdue.edu/flowcyt/software/Catalog.htm
Available analysis software
Free cytometry software:
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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
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• 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
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http://www.wfi.med.ic.ac.uk/Flow.htm
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