Guest Guest Posted March 29, 2023 Report Share Posted March 29, 2023 Hello, We are wondering if we can incubate PBMCs with our stimulus on the fluorospot plate for 6 or 7 days instead of the recommended time for each analyte or if there are any contraindications. Quote Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted March 30, 2023 Report Share Posted March 30, 2023 Hello there, We do not have experience incubating PBMC within the ELISpot/FluoroSpot plate for 6-7 days. I know this can be found in the litterature, but my suspicion is that results might come out rather weird. My suggestion would instead be to do "cultured ELISpot/FluoroSpot" using a cell culture plate. Pre-incubate the plates for 6-15 days in this cell culture plate with or without antigen and feed the culture IL-2 at certain days. Then, wash cells extensively and do an overnight incubation in the FluoroSpot plate at the end. I attach a PPT where we show this setup for ELISpot and how it compares to results generated using a direct ELISpot. As you can see, backgrounds go up but antigen specific responses are greatly improved. By lowering the cell number you achieve a much greater signal-to-noise for these antigens. A similar setup is certainly possible in FluoroSpot! Cultured IFNg ELISpot protocol.pptx Quote Link to comment Share on other sites More sharing options...
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