I derived M1 macrophage from human PBMC.Than I coated my CFSE stained thp1 target cells with my Abs on ice for 30mins, continue with 2 times washing in pbs----resuspend cells in IMDM (no serum)---meanwhile, wash mature macrophage with pbs for 2 times (macrophages are still adherent on 96 well plate)----add target cells in macrophage in 96 well (E/T=1:2)----co-culture for 2hrs in 37℃----discard supernatant that contain target cells----wash again with pbs for 2 times to make sure there are less and less CFSE stained thp1 target cells left in the well.----observe under immunofluorescent microscopy----detach the cells and stain macrophage with CD14 Ab, run flow.
Now my question is: When I observed under a microscopy, I found most of the macrophage have green fluorescence in them, even the same in the hu-IgG iso ctrl and Fc mutant Ab group.
Is it because the thp1 can very easy be phagocytose by macrophage? Or anything else I did wrong? Hope anyone can answer me. Thanks!
i also met the same problem. did you find the reason to figure it out? thx
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