PI/ Annexin V staining

Hi all

 

I have a question from a colleague. Please can you submit all replies
directly to her.

 

Thanks in advance

Sharon

 

To Purdue cytometry mailing list 

 

I am trying to use the PI/ Annexin V staining procedure to detect
apoptosis induced by infection of PHA stimulated PBMCs with various
HIV-1 isolates. In preparation I stimulate the PBMCs 3 days before
infection, and infect overnight. I wash the cell twice in 0.9% saline
(at room temp), resuspend in 100ul of 1x binding buffer then add 5ul of
annexin APC, leave at room temp in the dark for 15 mins then add PI
solution. Even after titering out the PI to very low concentrations I
see a very high proportion of PI+/annexin- cells in my cultures. 

I find very low levels early apoptotic (~1%), but more late apoptotic
(ann+/PI+, ~8-10%), and high levels of dead (PI+,ann-, ~20-30%).
However, instead of seeing a mobile band between early and late necrotic
cells, the cells appear to be shifting towards PI+/ann- during the
acquisition process. 

 

The literature seems confusing on this matter. If cells have died not by
apoptosis but either due to time in culture or shock during the staining
process (due either to temperature or osmotic changes) would you expect
to see them as PI+/Ann- ? 

 

Are there ways I can minimise necrosis during staining? also, has anyone
got experience on staining cultured PBMCs, and do they find a similar
high proportion of dead cells which do not stain with annexin? 

 

Thanks 

 

Polly Walker 

pollyw@nicd.ac.za