hello all - I'd like to do some Ca flux experiments in mouse splenocytes. It looks like loading with indo-1 is the way to go, and stimulating with anti-CD3e clone 145-2C11. Does anyone have a protocol they're willing to share? In the literature I've seen indo-1 between 1 and 5 uM, and anti-CD3 between 1 and 90 ug/ml. I'm hoping to do direct ex vivo studies on CD8 cells. We're thinking to stimulate them to flux by addition of MHC I tetramer and sort by 62L expression (using 4 color flow). Can anyone give advice as to what anti-CD8 or anti-CD62L clones WON'T stimulate Ca flux? And finally, has anybody tried this sort of thing on PBMCs? Thanks a bunch Shawn Jackson Div. Viral Pathogenesis Beth Israel Deaconess Medical Center Boston, MA 02115 617-667-0182
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