Annexin V and PI or 7-AAD protocol

2014-08-29

azim58 - 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

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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.

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