Hi, I have incubated my anti-CD19 CAR-T cells with CD19 coated beads (upped to 5 beads for 1 cell in my last experiment, transduction rate 30%), 3 hours later I have added protein export inhibitor (BFA+monensin). I get 0 IFNg and TNFa staining after overnight culture (my positive control is PMA/iono and I get a beautiful staining).

My objective is to use beads to activate my CAR-T cells, so checking for intracellular cytokine production seemed legit (my cells are 100% CD25+ and sometimes up to 60% CD69+). I could check for cytokines released in the medium, but I will have no idea what %age of CAR-T cells got activated. I need max.

BUT, I am guessing there is very high chance it will never work and BFA+monensin are blocking the activation of cells through CAR (BFA+monensin are blocking the export of CAR to the surface, I lose 10% of staining).

Has anyone tried such an experiment? If intracellular stainign never works, should I just check HLA-DR upregualation ?

How would you do CAR activation?

Thank you !!!!