Guest Tim Posted February 7 Report Share Posted February 7 Hi, We are observing strange and reoccuring spot counts in wells with only PBMCs used as negative control from one donor in IFNg ELISpot. We do not see this effect with cells from other donors, but we are wondering if this donor might just be very sensitive to possible contaminations. The spots are always inhomogenously spread in the wells and are not present in all wells although the cells come from one vial. Maybe someone here has an idea? Thanks in advance! F3.bmp F3.bmp F3.bmp E11.bmp D12.bmp Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted February 7 Report Share Posted February 7 Hello! IFNgamma will have background in ELISpot for some donors. If you test 20 donors, around 1-3 will show up with elevated levels of IFNg secretion in wells where cells have only been incubated in cell culture medium (ie my definition of background). If you have contaminated your medium or plate you risk ofcourse having even more backgrounds. This is also the case when testing certain patient populations where the invivo immune system is inflammed, resulting in T-cells being more prone to IFNg backgrounds. When looking at your images I would certainly describe that has very high IFNg backgrounds for healthy donors. Maybe this is a patient of some sorts? The distribution of the spots will be affected by pipetting technique. Also if you first add cells and afterwards add medium with or without stimuli, cells will be pushed to one side of the well. Always best to first add media, then cells. Quote Link to comment Share on other sites More sharing options...
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