Hi,
I am checking the antiinflammatory effect of few substances on a co-culture model. I have screened the pro-inflammatory cytokines with ELISA kits.
Do I need to confirm the result with another method?
If yes which method/assay is the best?
Regards,
If you saw by ELISA that your antiinflammatory substances reduced the measured concentrations of pro-inflammatory cytokines in the extracellular environment, and granted that you have appropriate negative and positive controls, this is already quite good.
If you want to firmly demonstrate that your antiinflammatory substances have a selective impact on pro-inflammatory cytokines, then you may need to use a multiplex assay (such as Bioplex) to measure many factors at once.
If you want to add another figure, you could of course perform qPCR, but the informative value is of course not as good as what you already saw by ELISA and you may need a higher cell quantity.
If your cell population is heterogenous, you could use flow cytometry to identify the exact cell population that is impacted by your treatment (however not all cytokines are easily detectable by flow cytometry). If your cell population is tissue-related you could also stain tissue sections to look not only at what population produces the inflammatory cytokines of interest, but also where they are located and with which cells they interact.
Finally, you could also consider (if you did not already) performing functional assays by adressing the effect of your supernatants on cells to prove that the antiinflammatory sustances not only modify the release of proinflammatory cytokines but that in addition it has an impact on target cells.
Thank you so much for your answer Julien.
Do you think western blot can be used as a confirmatory?
Sure, this is actually an excellent idea.
Western-blot would not really be useful to confirm your results stricto sensu, since quantification is way less accurate than with ELISA but you could address whether some of your cytokines of interest had post-translational modifications (e.g. phosphorylations).
The interesting thing to do, in my opinion, would rather be to determine if your antiinflammatory substances affect various intracellular pathways such as MAP kinases, NF-kappaB etc in your treated cells. This would not be a confirmation that your substances have an impact on production of proinflammatory cytokines, but this would be an explanation for this and this is actually even better in a way.
I agree with Julien that, using appropriate negative and positive controls, ELISA should be enough to demonstrate differences in expression levels of cytokine. Anyway, if you wish to carry on further ancillary experiments, you could quantify also mRNA of target cytokines by qRT-PCR.
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