I am doing ICS with human PBMCs to look at IFNg production from CD8 after stimulation with autoantigen epitopes. I have a few questions:
1) How long do you usually stimulate the PBMCs with the peptides? I know it must be very different depending on how abundant the particular clone of CD8 is present, but I would like to hear some experiences.
2) Should I incubate the culture with brefeldin A right from the start or the last 5 hours of the stimulation.
3) I am including positive controls for IFNg, both PMA/ionomycin and peptide positive controls (FLU/EBV/CMV). Should these be added to the PBMC at the last 5~6 hours instead of same time as other test peptides to avoid over-stimulation/apoptosis (if peptide stimulation do needs more than 5~6 hours stimulation)? I know I should keep the incubation time all the same, what do you do to overcome this?
There are two issues: PBMC incubation with peptide to expand peptide-specific T cells (in vitro restimulation) and peptide stimulation to detect antigen-specific IFN production. For the in vitro restimulation: usually you have to perform one or two (sometimes more) rounds of in vitro restimulation. For each round, wait 5 days after peptide addition to allow for i) T cell proliferation and ii) overcome a period during which T cell are desensitized. There are many different protocole of weekly in vitro restimulation for T cells. For the IFNg assay itself, add your peptide or your positive control peptide or your positive control PMA/iono to T cells at time 0 then incubate for 2 hours. That's the right moment to add brefeldin A. Then incubate for 3 more hours before to wash cells, perform surface staining then permeabilize your cells and perform intracellualr staining.
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