I'm using venous blood drawn into sodium citrate, then lyse RBCs with ACK lysis buffer. The amount of blood is usually around 100 uL. Doing 4-5 hours stimulation with PMA-i, adding BfA 1 hour post stimulation start. Then I follow common procedure for surface markers followed by intracellular staining for flow cytometry. The issue is that I always get very poor TNF stimulation, while IFNg stimulation is not perfect, but still detectable. The whole procedure is done with a single purpose of confirming IFNg or TNF knockout in my pups.

I've tried to look how I can improve the protocol, but couldn't find any decent published protocols. Can please anyone help with any advice on how to improve TNF stimulation in mouse PBMCs from venous blood?