This issue was discussed a while ago. Someone suggested to use calcium after isolation of adherent cells using EDTA. When we used calcium containing buffer to wash the cells after EDTA-Trypsin treatment, we saw reduction in apoptosis frequency in an adherent cell line. We substituted regular Dulbecco's PBS (GIBCO-BRL) with CaCl2 at a final concentration of 1mM. We washed the cells once before apoptosis assay. It has considerably reduced the background. You may want to try this. Good luck Jay > -----Original Message----- > From: Anke Kretz [SMTP:kretz-rommel@alxnsd.com] > Sent: Friday, May 18, 2001 4:16 PM > To: Cytometry Mailing List > Subject: apoptosis assay on adherent cells > > > I am trying to determine apoptosis induction on adherent tumor cells. I > used annexin/PI staining, however the background of annexin staining in > untreated cells was up to 20 %. I am concerned that the cell > dissociation buffer already induces apoptosis. I also tried IHC, but > was loosing a lot of cells when I was trying to fix them. Does anybody > have a good protocol how to do apoptosis measurements in adherent cells? > > thank you, > Anke
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