Problems with AnnexinV-FITC staining - (Sep/14/2006 )

Hi

I am Sweta *
a new member of this forum. I am working with detection of early apoptosis by AnnexinV-FITC staining pattern determined by Flow cytometry.
For this, I use staurosporin as an apoptotic inducer, trypsinize cells for 3 mins, washed cells with PBS twice to take the cells into eppendorf tube, centrifuge the cells , resuspend the pellet with 1x Binding buffer, then I add annexinV-FITC and PI/7AAD, incubate in the dark for 15 mins , then do flow analysis.

But I find more AnnexinV-FITC positive cells even in the controls cells without any treatment. If I incubate the cells for more without serum the percentage of annexin positive cells increse accordingly, although they are not undergoing apoptosis or necrosis.

So I dont know what is the problem, why these cells are positive, is it something with the cells o with the antibody (BD bioscience)


I am using BD annexix FITC-apoptosis kit(1xBinding buffer, AnnexinV-FITC, PI} for the experiment.

Please let me know, how can I get rid of this problem, I mean how can reduce the background staining
i would be grateful to you.
regards,
Sweta

Maybe you can look under a fluorescent microscope to see if it's true that more unthreated cells are positieve or your FACS setting might be wrong.
I don't know much about the FACS, I always count the cells under the microscope. I never had problems with these kind of stainings.

I think this types of problrms due to trypsinization. You can replac trypsinization by detaching cells
pipetting any other methodes like using scraper for detachment of cells. Second things keep in mind gives less stress to the cells during prossesing like not spun at high rpm and staing volume of buffure not will be more than 50-100 ul.