FACS issue for Apoptosis?! Propidium Iodide Vs Annexin V-Cy5

[pjberlin]

Questions regarding establishment of FACS method to study Apoptosis upon cytotoxin treatment.


I want to Discriminate Live, Early and Late Apoptotic and Dead cells post treatment with a particular cytotoxic agent and analyze using FACS (BD FACS CANTO 2).

Propidium Iodide Vs Annexin V - Cy5

1. I am using 2 step Annexin V detection (We dont have Annexin V-FITC). Will this work
effectively compared to Annexin-FITC.

(rabbit anti-Annexin V antibody for 10 min, WASH,Goat Anti-rabbit conjugated to Cy5 for 10 min)

2. Will this give best results to discriminate different apoptotic phases.

- 2ul Rabbit anti-Annexin V antibody for 10 min; WASH in 1mLPBS-BSA-EDTA, Centrifuge 4min
- 3ul Goat Anti-rabbit conjugated to Cy5 for 10 min; WASH in PBS-BSA-EDTA,Centrifuge 4min
- 4ul Propidium Iodide for 10 Min, WASH in PBS-BSA-EDTA,Centrifuge 4min
- Resuspend in 400ul PBS-BSA-EDTA.
- Analyze

3. Best working Propidium Iodide solution provider for FACS? (I am using BD Pharmigen, 4ul for
10^5 cells). But it doesnt seem to work well. I could not see the shift of untreated cells
towards PI channel when stained with PI alone.


Hope this resourceful scientific world assist this new comer to FACS!

PJ