Can anyone explain why a study's ELISpot assays might be designed wherein its cells are being plated at a concentration of 200K/well (the standard amount) and then 100K/well? The concentration of the peptide being tested does not change, just the cells. We are somewhat blinded to what the study sponors are looking for, so I don't have the full details, but I struggle to understand what the point of this might be. Would a lesser concentration of cells be informative of the efficacy of the peptides? I would think that playing with the test peptide concentration would be better than that.
Hello Benjamin Justin Mildenberg
The number of cells must be adapted to the cell type as well as the frequency of secreting cells namely, the lower the expected frequency of responding cells, the higher the number of cells per well. For instance, if the expected frequency is 1 in 10,000 cells, then you will need approximately 200K cells per well. Conversely, when the expected frequency is high, say 1 in 100 cells, then it will be sufficient to seed 100K cells per well.
Another important factor I would like to mention is that antigen specific responses will require higher cell number (200K) while the use of mitogens may require lower cell number (100K). You also need to know that a minimum number of cells is required for proper cell-to-cell contact and thus for optimal stimulation. For example, if one is analyzing antigen-specific T cells in a PBMC sample, the cell number should be 200K cells per well.
So, cell number would play an important role in ELISpot assays in addition to the test peptide concentration.
Best.
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