I used to prepare peptide/BFA cocktail and add to resuspend spleen cells, so that means the stimulation and cytokines accumulation simultaneously. Currently, I read a paper on PLOS ONE (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038926) that talk about differential stimulation and accumulation approaches alter the cytokines profiles. I just curious about you guys how to process this experiment.
Based on my observations and experiences, peptide/BFA stimulate cell 4-6hr looks normal protocol and has been reported in plenty of papers, the other one is peptide stimulation overnight, and accumulate the cytokine by adding BFA for 4-6hr.
I wonder which one you guys are preferring. Thanks.
Hey Hou,
To me, both these protocols work well for intracellular cytokine staining, although I preferred the latter one, i.e. first adding the lymphocyte stimulators (such as PMA and ionomycin) and then BFA/monensin. You can see my paper (Shekhar et al., J Innate Immun, 2015) if you wish.
Good luck,
Sudhanshu
You welcome, Hou! Here is the link you asked for: https://www.researchgate.net/publication/269407602_Invariant_Natural_Killer_T_Cells_Promote_T_Cell_Immunity_by_Modulating_the_Function_of_Lung_Dendritic_Cells_during_Chlamydia_pneumoniae_Infection
Article Invariant Natural Killer T Cells Promote T Cell Immunity by ...
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