Does anybody know how to normalize the level of a cytokine to the number of cells treated with a drug? The point is that you have a huge reduction in cytokine production but have to exclude the effect of cell death. I can share more details if needed.
Something you can do is to check the viability of the cells in parallel at different time points and at the end of the assay, for instance by using a MTT assay. Knowing the initial number of cells cultured and the % of viability for the time points you will have a reference of the number of cells under analysis.
If accelerated apoptosis or cytotoxicity is not the mode of action of the drug being investigated, you may also use ELISPOT for both quantitative and qualitative evaluation of the treatment effects.
Intracellular immunostaining suggested by Laure Perrin-Cocon is helpful and you can screen for dead cells, except that you don't know how much of it is released from the cells and how these cells contribute to the level of cytokines in the extracellular microenvironment. A combination of ELISPOT, Luminex assay, and Flow Cytometry (intracellular immunostaining) will be best.
You can also normalize cytokine/chemokine concentration in supernatants to total protein content (measured for instance with the BCA assay) in adherent cells after supernatant collection.
Dear friends:
I really appreciate all the nice and helpful suggestions. I could succeed with a combination of viability assay, ELISA, and intracellular cytokine staining (FACS)!!!.
Many thanks
I use CCK8 kit to measure cell viability. Its fairly accurate and straightforward to use. Also non toxic. Can be used along with cytokines.
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