Hi
I use A549 cells and stain them with Annexin V-FITC and 7-AAD.
I induce apoptosis with staurosporine and UVC as positiver control but I don't get appropriate amounts of apoptotic cells ! max is ~10%, and always >80% still living!

I wonder if the trypsin I use to detach the cells somehow alters the cells, so the Annexin cannot bind to the phosphatidylserine anymore..?

Maybe anyone got an idea.. or if anybody got a working protocol for A549 cells and AnnexinV / PI - staining (7-AAD respectively).. I would appreciate that.
What agent do you use as positive control and how much apoptotic cells you got?

Thanks
Frosti

Hey did you find anything??
If so, appreciate it if you could share the protocol with me.
Thanks
P..

I don't do this assay but I do detach my cells for other applications with EDTA/PBS first then a few minutes later I add collagenase. Cells detach beautifully. If you want details let me know and I will find out the concentrations.

Hi MaximinaNYC,
What does the collagenase do?? The prob is that the EDTA-trypsin detachemnt seems to be brutal to my cells and everything seems to be taking up annexin. Scraping also does not work. I have pretty much given up on the assay.

If you dont mind please me the protocol for the collagenase thing...let me see how it works. Thanks a ton.
Regards,
p...