Dear Colleagues,
I need your opinion on explaining one phenomenon we observed in mouse IFNg-Elispot assay. We desire to test Ag-specifc T cell responses by IFNg-Elispot assay. To do so, we incubated the cells with peptide pools and with medium alone as negative control in addition to our normal control mouse group. In general, we expect to see no or minimal number of spot signal in the medium well. However, from time to time, we observed great number of spots (10-100/well) in the medium well. I do have some speculation on this result, but your comments or sharing your experience will help us to better approach the issue.
Thank you very much!
Haifeng
Thank you, Niklas for the input. When you say non-specific activation, you mean T cells? Do you mean those cells constitutively secrete IFNg? I speculated the possibility that some Ag-specific T cells were so robustly activated that they don't need more ex vivo stimulation to secrete cytokines although a proportion of the cells may be non-specific ones. I also wonder if there are NK cells there too.
As you suggested, we may want to test it at more time points to see how it goes.
Thank you again,
Haifeng
IFNg can also be secreted by natural killer cells and natural killer T cells as part of innate immune response. Are spots in treatment and control group are statistically different ? Have you restimulating cells?
@Mesula, I agree. We use peptide pools to stimulate the cells and with medium alone as negative control. We saw difference between these two conditions but haven't done statistical analysis. We will design panels to see NK cell response. Thank you!
It seems that the cells you incubated without any stimulant or peptide etc (negative control) are responding to something you are not controlling for. For instance, it could be that the animal had been exposed to some pathogen, or your cells are contaminated etc. Or it could be that you just have high background noise. This happened to me before which makes it hard to evaluate specific response against a particular antigen. You can calculate relative CFU counts per well by subtracting the background.
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