Hi Mabtech,
I was wondering if you have any experience on the minimum cell number to use for an IFNg/IL-17 flurospot assay? I am planning to sort cells and will have very limited cell numbers (in the single thousands range). I plan to stimulate the cells in various ways (all polyclonal stimulation) and it would be very useful with some input if this approach is even feasible in the context of fluorospot?
Thanks for your always very helpful insights,
Emelie
T-cell analysis in ELISpot/FluoroSpot is to a certain extent dependent on cell-to-cell contact, even for some polyclonal stimuli.
PHA works fine at 50,000 pbmc/well for IFNg, but can drop to almost nothing at 20,000-25,000 PBMC/well. This can vary from donor to donor but it is important to understand that it is not totally linear. The same is true for the anti-human CD3 stimuli from Mabtech. It can induce IFNg very reliably at 50,000 cells/well but below 25,000 you suddenly go off a cliff.
By contrast, PMA+Ionomycin is much more independent of cell-to-cell contact and will stimulate T-cells great even at very low numbers of cells/well. I think I have seen people use just 5000 PBMC per well and it worked. PMA+inomycin can be tricky since it is a light sensitive stimuli and I recommend to always prepare it fresh before use.
best,
Christian
Thank you so much for the reply, then there is hope- I'll give it a try and evaluate using different stimulus. Do you by the way have any advice on how to only use parts of the plate for a pilot experiment? Can this even be done considering that the whole plate is put into the incubator?
Best,
Emelie