Annexin+V+and+PI+or+7-AAD+protocol


 * Annexin V and PI or 7-AAD protocol**

__Materials__
 * Binding buffer
 * Annexin-V APC

Before starting: Dilute 10X binding buffer to 1X using distilled water (1mL 10X binding buffer + 9mL dH20)

1. Wash cells once in PBS 2. Wash cells once in 1X binding buffer (prepared above) 3. Resuspend cells in 1X binding buffer at 1-5x10^6/mL 4. Add 5uL of fluorochrome-conjugated Annexin V to 100uL of the cell supension 5. Incubate 10-15 minutes at room temperature in the dark 6. Wash cells in 1X binding buffer 7. Resuspend in 200uL of 1X binding buffer 8. Add 5uL of PI staining solution OR 7-AAD staining solution. 9. Analyze by flow cytometry within 4 hours, storing at 2-8C in the dark

Note that to run through our flow cytometer, should add ~700uL of 1X PBS right before analysis so that the final volume is 1mL (as preferred by the cytometer)

Documents

Interpretation of Annexin-V results: Annexin V-negative and viability dye (like 7-AAD or PI) positive indicates late-stage apoptotic and necrotic cells. On the other hand, Annexin V positive and viability dye negative indicates early-stage apoptotic cells.