Hello everyone, I am quite new in Elispot assay. I am trying to develop a human IFN-gamma Elispot assay for human PBMCs which co-cultured with adherent tumor cells. I tried to search a protocol but could not find a good one. Since the tumor that I am using are adherent I cannot develop Elispot with PBMC-tumor coculture. Is there anyone who could give me some tips? Thank you.
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IFN-gamma Elispot with human PBMC activated by adherent tumor cells
- GEGuest Esen Aral2024-02-22T16:14:13Z
- CChristian@mabtech.com2024-02-22T16:33:11ZAdmin
Hey and welcome to the Forum!
Many times the usage of adherent cells in ELISpot is not that huge of a problem. It can be, but not always.
One reason for this is that the spots you can see with your eyes are made of billions of cytokine molecules and rather large, whereas adherent cells not washed away by the PBS washes will not interfere too much with the substrate development process.
At the same time, If you find that they do strongly adhere to the membrane and appear to interfere with the IFNg spots, you can add this 1mM EDTA step right after the cells have been washed away (first wash after incubation). Add in the 1mM EDTA in all wells (100ul/well) and place the plate for 10min in 37C. Afterwards you wash again 5x PBS and continue normal with the rest of the detection process.
The short treatment with EDTA at 37C will help adherent cells disslodge from the PVDF membrane.