Guest Sap Posted January 16, 2019 Report Share Posted January 16, 2019 Hi, I have been standardizing ELISPOT assays in our lab with dendritic cells for IL-12p70 and IL-23 using your Pro kits. I did not see any spot formation for the 1st set of the experiment for 24hr incubation time. Do we require to incubate the cells longer 48hr or 72hr to see any spots for these cytokines? What are the kinetics of these cytokines? Do I have to do extra washes or anything if I go for such long incubation hours? Also, I am using LPS and IFNg as my positive control. Does LPS require serum in the media to work? I have been using serum-free media for my ELISPOT experiments. Please advice. Thank you, Sap Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted January 16, 2019 Report Share Posted January 16, 2019 Dear Sap, Welcome to the forum. I have some experience working with dendritic cells in ELISpot/FluoroSpot. How are you generating your DCs? IL-4+GM-CSF for 3 days? Is the viability good before you add them to the ELISpot plates? What cell numbers/well are you using? In my experience R848 is a very good stimulating agent for dendritic cells. On the other hand LPS+IFNg should work good as well. Overnight incubation should be enough. Tomorrow I can post some experiments i did years ago with DCs... Link to comment Share on other sites More sharing options...
Guest Sap Posted January 17, 2019 Report Share Posted January 17, 2019 Thank you for your reply Christian. Yes, DCs have been generated by IL-4+GM-CSF. The viability is not that good its around 40-50%. I know we should have 80-90% viability but I cannot help it here! I have to use these cells and do the ELISPOT. I have been trying two cell dilutions 500,000 cells/ well and 250,000 cells/well. I do not get anything in 24hr incubation. I have been adding LPS and IFNg together with the cells. Is that enough? Link to comment Share on other sites More sharing options...
Christian@mabtech.com Posted January 17, 2019 Report Share Posted January 17, 2019 Hi Sap! Ok so your experiment was with monocyte derived DCs. Viability is not great. In my experience anything below 60% and you run the possibility of getting zero result. The dead cells influence the living cells and causes them to secrete almost no cytokine. But I have to say, my experience comes from T-cells here, not DCs. Why is you viability so low? What is your source of PBMCs? For how many days do you incubate with GM-CSF+IL-4? I looked into my old experiments and please have look in my attached PPT. I here used FluoroSpot but results in ELISpot would be very similar. I did not test IL-12p70 but I did evaluate IL-23. At 10,000 cells per well, spot numbers were low, even with the strongest stimulant (a TLR7/8 ligand called 3M019). Maybe you can test TNFa and IL-12p40 instead? Both of these products are available in our webshop. The IL-12p40 combines IL-23 and IL-12p70 secretion. How can IL-12p40 be so high be so high in terms of spots in my ppt? Well this system also detect monomer secretion of p40 and this is probably secreted at rather high levels by DCs. Atleast that is my speculation. Please note that TNF-a results have been obtained with only 1000 DC/well. Overall this is a very good cytokine to include as a "positive control" system MDDCs results fluorospot.pptx Link to comment Share on other sites More sharing options...
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