Just after isiolation of any cell I suppose the population is under the stressful situation during in vitro study, it is therefore suggested that even stimulation should be after an hour and the treatment should be after a gap of at least 6 hrs, however optimization of your own protocol matter.

Hi Santhuri, I believe that your problem is not the time of BFA addition after stimulation, the problem is the time of incubation after BFA. You are supposed to add BFA to your cultures for the last 4 hours of incubation. If BFA remains for over 6h the cell loss is significant. The cells die. Monocytes produce detectable levels of TNFa and IFNg 6 h post-stimulation, but you can also detect good numbers of cytokine-producing cells at 18-24 h. I'm not sure about IFNa. Consider that monocytes are a small portion of the total PBMCs and that you should be acquiring at least 100,000 cell to be able to detect specific responses.

Good luck with your experiments.