Protocol for assessing apoptosis by Annexin V binding :
1. Harvest cells, wash twice in cooled PBS and resuspend at a concentration
of 1 x 106 cells/ml in Binding Buffer.
2. Aliquot cells (100 ul) into FACS tubes and add Annexin V and/or viability dye.
3. Mix gently
and incubate for 15 min at room temperature in the dark
4. Add 400 ul of Binding Buffer to each tube and analyze immediately by flow
cytometry.
Note: The samples can be kept at minus 20 centigrade for 4-8 weeks.
Recommended tubes:
(1) cells
alone
(2) cells + Annexin
(3) cells + PI (or 7-AAD)
(4) cells + Annexin + PI (or 7-AAD)
Annexin V is available
in biotin, FITC and PE formats. When using Annexin-V-FITC, PI can be used as the viability marker (5 ul of a 50 ug/ml stock solution).
When using Annexin-V-PE, 7-AAD may be the preferred viability marker (1 ug/ml final concentration) as there is less spectral overlap
of PE and 7-AAD than PE and PI.
REAGENTS:
Annexin-V-FITC
Annexin-PE
Binding Buffer 10X, (0.1M HEPES/NaOH, pH7.4; 140mM
NaCL; 25mM CaCl2),
Propidium Iodide
7-AminoActinomycin D
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Ch 8.Immunohistoch / immunology
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