Apoptosis detection with Phosphatidylserine antibody - (Mar/06/2008 )
Hi all,
I am performing experiment to detect near apoptotic cells using FITC labelled phosphatidylserine antibody bought from Upstate. I am using Hydrogen peroxide in media to induce apoptosis like stress condition. I treat cells with hydrogen peroxide for 1 hr in 24 well plate. Then I wash them with PBS and incubate for another 1 hr with FITC-anti-PS antibody(diluted in cold PBS) on ice bath. Then i measure fluorescence intensity before and after washing with cold PBS using fluorescence plate reader. Now, the problem: before washing I get expected results for blank, control and treated but after washing i donot detect any antibody binding to the treated cells (i almost get the same readings). I have performed this experiment for four cell lines. Is the problem that I am using following two unknown parameter here?
1. If or not hydrogen peroxide is able externalize Phosphatidylserine in cells.
2. If or not antibody is working.
Please help me in whatever way you can. Thanks.
I did this staining methods for a while ago, but I used flow cytometer not plate reader,
anyway, since you are getting expected result before washing cell, I think antibody and assay are working,
problem should be washing methods.
I am performing experiment to detect near apoptotic cells using FITC labelled phosphatidylserine antibody bought from Upstate. I am using Hydrogen peroxide in media to induce apoptosis like stress condition. I treat cells with hydrogen peroxide for 1 hr in 24 well plate. Then I wash them with PBS and incubate for another 1 hr with FITC-anti-PS antibody(diluted in cold PBS) on ice bath. Then i measure fluorescence intensity before and after washing with cold PBS using fluorescence plate reader. Now, the problem: before washing I get expected results for blank, control and treated but after washing i donot detect any antibody binding to the treated cells (i almost get the same readings). I have performed this experiment for four cell lines. Is the problem that I am using following two unknown parameter here?
1. If or not hydrogen peroxide is able externalize Phosphatidylserine in cells.
2. If or not antibody is working.
Please help me in whatever way you can. Thanks.
Thanks. Did u get good results with Flow cytometer? And how did you detached the cells?
What do you think might be wrong with my washing method coz i use PBS. Pl. reply.
anyway, since you are getting expected result before washing cell, I think antibody and assay are working,
problem should be washing methods.
I am performing experiment to detect near apoptotic cells using FITC labelled phosphatidylserine antibody bought from Upstate. I am using Hydrogen peroxide in media to induce apoptosis like stress condition. I treat cells with hydrogen peroxide for 1 hr in 24 well plate. Then I wash them with PBS and incubate for another 1 hr with FITC-anti-PS antibody(diluted in cold PBS) on ice bath. Then i measure fluorescence intensity before and after washing with cold PBS using fluorescence plate reader. Now, the problem: before washing I get expected results for blank, control and treated but after washing i donot detect any antibody binding to the treated cells (i almost get the same readings). I have performed this experiment for four cell lines. Is the problem that I am using following two unknown parameter here?
1. If or not hydrogen peroxide is able externalize Phosphatidylserine in cells.
2. If or not antibody is working.
Please help me in whatever way you can. Thanks.
Have you looked at the cells under a microscope first? All you questions should be answered by yourself with your next experiment, not by us remotely.
well, for your questions frist,
my annexin/Pi staing with flow cytometry worked very well, and since I used leukemic cell lines (suspension cell), I did not use anything to detach them.
for your wash, PBS should not do anything for this stainig, so I dont know exactly what causing the situation.
maybe your wash somehow wash all cell away from plate and you are looseing cell??