i wanna perform CAR T cell mediated cytotoxicity assay by using CFSC/PI. As PI cannot cross the membrane of live cells, making it useful to differentiate necrotic, apoptotic and healthy cells. i wanna include one positive group for PI, in which PI can cross the cell membrane and give me high % of positive PI cells upon FACs analysis.
How should induce necrosis or apoptosis in my healthy cells, so that they stained highly positive for PI staining ??
I am using MT-2 cells, before i tried with multiple boiling(60c) and freezing (-80), but it didn't work.
Thanks.
Hi,
There are many studies which suggest to use Hydrogen peroxide and Lipopolysaccharide (LPS) for the induction of apoptosis and necrosis respectively.
I myself use Hydrogen peroxide for the induction of apoptosis in breast cancer cells.
Usually, two cycles of freeze (-80) thawing (37) kills most cells I’ve worked with. Since that’s not working for you, you could fix and permeabilze the cells.
Dear Sumit Goel Allison C Sharrow Han Zhang Thanks for you valuable suggestions. I recently perform the assay, and i tried Triton X100, as it is commonly available in our lab. Its work very well, upon 10mints incubation period with Triton X100, i gate 90% positive cells for PI.
Thanks.
Thank you! We ended up usibg 0.5% Triton for 30 minutes and jt seem to work well.
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