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Guest Esther

Stimulation with target cells (co-cultures)

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Guest Esther

Hej,

I want to use the IRIS to check for cytokine production by T cells in response to their target cancer cells.

The cancer cells are transduced with the target antigen and our cells are highly responsive to them.

Are there protocols available for how to set up co-cultures for the IRIS and is it possible to use adherent cells?

Best, Esther

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Hej Esther!


Co-cultures of e.g. cancer cells and effector cells is a quite common setup, although we hardly ever perform these experiments in-house. The important thing is that you find a suitable target:effector ratio and to include control wells with e.g. only the target cells to make sure that they don’t produce the cytokines of interest themselves. For protocol inspiration, you can have a look in this paper by Zuber et al 2005: https://www.ncbi.nlm.nih.gov/pubmed/16005014.


Although we do not have extensive experience of using adherent cells in ELISpot/FluoroSpot we have not encountered any problems in the few experiments we indeed have made. The cell types we have analyzed include different epithelial cell lines where we have either looked at the production of cytokines (e.g. IL-6 and TNF-alfa) by these cells or have used these cells as APCs. When looking at things secreted by epithelial cells you actually get very nice spots looking very much like spots produced by lymphocytes and there seems to be no interference from possibly remaining cells sticking to the membrane.


To my knowledge, there is no special protocol for using adherent cells in ELISpot. We have just used our regular protocols provided in the kits. However, the risk I think one may anticipate when using adherent cells is that they will stick more firmly to the membrane and by not being washed off properly will cause background problems. As said, we have not changed the washing protocol for in our in-house experiments with adherent cells, but logically you could think that some more harsh washing procedures would be required. We normally wash with only PBS in an automatic ELISA washer but one could consider using PBS with 1 mM EDTA and leaving it for some time in the well (most adherent cells would detach under these conditions). I know that some groups also include Tween in their washing buffer but we have never seen any beneficial effect of this but rather that you may sometimes see increased background.


Hope you at least got some ideas! Don’t hesitate to come back if you have further questions.

Kind regards,
Jens

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