I'd like to know if it would be better to increase the number of cells plated or increase the peptide concentration? Which one would result in a better increase in spot formation.
ELISpot assays have a very well defined linear range for cell number for mouse/human IFNγ ELISpots the cell# to spot# ratio is linear from ~200-800 thousand cells. Any more than that won't help (and will in fact hurt). Peptide concentration can certainly help. T cells have a sigmoidal dose response to peptide-MHC. Most peptides are approach saturation at around 10 ug/mL. At times higher concentrations are need, but that is typically not the case. Are your spots intense, but present at low numbers? What are your "low" spot numbers that you want to increase? Do you have background spots?
Currently I plate 200k cells. I've never really run these assays before so I don't have much to compare to. I believe my spots are fairly sharp, but in low numbers. I do have some background spots.
It's common to have < 50 antigen-specific spots/million cells, so low spot numbers may be expected with 200K cells. You should definitely increase cell number and expect an increase in spot number. Do you use an automated spot counter? Do you gate out background spots based on spot size?
Typically we use 10ug/mL peptide as above as a 'saturating' dose, so I agree with Adam. I like 1x10^6 cells per well because it is clean, but up to 10x10^6 will work well for an IFN-g ELISpot. As a positive control (peptide + antiCD3), even 0.5x10^5 cells will give almost an entire well of spots, so it does somewhat depend on the peptide you use.
You may also consider changing your detection and development conditions to adjust for sensitivity
Working with only 250uL of blood so I'm limited in the # of cells I can plate per sample. This is my dilemma.
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