Problem with Annexin FITC antibody - (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

Hi Shweta,
Here is some problems in your protocoles i thinks. You shuld avoid trypsinization of cells you can used detaching cells by pipetting with 1ml pippets.
AS you know annexine binding to exposed surface of PS. And trypsin not recomended for cell surface study.
I thinks you should try its for atleast one more time but keep in mind as much possible gives less stress to the cells during prossing by avaioding excessive higher speed spining and roughly handeling during detaching cells.
I last year facing same problems like you.
but now geting good results.
all the best.
awadh

Maybe you want to neutralize trypsin with 1 volume of 10% FBS-medioum or trypsin inhibitor before washing?

Hi Shweta ,
you not told which cells you are using. I thinks after completing all procedure you follow following sugesstion
-
removed all media and add 1xPBS and detached cells by pipeting or using scraper for detaching cells and take in eppendoff tube.
-repeat this steps by adding again 500ul of 1xPBA and keep in mind cells place on the ice during procedure
- then now follow all procedure discribed in your staining kit provided by manufacturer
but keep in mind one things gives less strees to the cells during process.
I think now you understood its.
any way for further quiary you can also contact directally without any hesitation on my id awadh507@gmail.com
all the best and good luck
awadh

I suppose it depends on the cell type, but I trypsinize my cells for 5 minutes, and still get very good results by annexin-FITC. The quenching might be important though - I agree with genehunter that neutralizing the trypsin is a good idea.

In our lab, we sometimes get annexin-positive cells in our controls if we let them get too overconfluent. Could that be a problem for you?

Good luck.