Hello.
How exactly does Con A work for mouse IFN-g ELISPOT? I understand it's a mitogen and non-specifically stimulates T cells. But does it stimulate ALL T cells (naive vs effector vs memory)?
Basically, I've seen ELISPOT data from a graduate student in the lab which suggests that the dosage for best IFN-g immune response observed for a particular adjuvant is 5 ug (approximately 5-10x greater than other dosages). Data seem convincing, BUT the Con A for 5 ug group is also 5-10x greater than other dosages. I had asked her could it be that her cell count was off for the 5 ug group and so she got 5-10x greater response because she added 5-10x more cells into those wells. She's adamant that her cell count was fine and that Con A is used to just make sure the ELISPOT worked and not to "normalize" for anything. She says that the reason there is 5-10x greater spots in Con A for 5 ug group is just because 5 ug adjuvant had the "best" response and therefore, there are more T cells in the spleen of that group.
Is she right? In my experience, I've never seen data where the Con A differed that drastically between groups. If Con A has no "purpose" besides to make sure an assay worked, then is it even necessary to do Con A for all groups within a given expt? Couldn't I just pick a random group and do Con A for just that group?
Thanks in advance.
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