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PBMC proliferation not working!!! - (Jun/09/2011 )

Hi every Body,
I've trying for some time to evaluate the proliferation of Human PBMC with H3... but the cells are refusing to be activated, i've tested the the Anti CD3- Anti CD28 coated beads from Milteny, i've tried to add the anti CD3 and anti CD28 to the milieu, i've tried the PHA, but nothing is working!!!! the H3 is being added after 24H in the incubator for another 18 hours. I'm Using the RPMI + 10% Serum AB.
thx a lot if u got any ideas that might help



)H3-thy assay is junk. Use cell cycle and CFSE analysis by flow

-Denis Baev-