I have antibody against a particular cytokine. I also have that particular cytokine. I want to detect that cytokine if it is released by the cells in the media. How can I prepare the sample using media so that I can detect the cytokine using antibody? I even doubt whether western blotting is a suitable method to detect cytokines in the media. Please troubleshoot and suggest some experiment plan.
Thanks
- Grow your cells in its normal media until desired confluency
- Next day, lightly rinse your monolayer with warm PBS. Add fresh media. If you gave some kind of drug treatment, treat your cells overnight with it (if not skip this step)
- Stimulate your cells with some kind of stress, for example 1mM hydrogen peroxide for 30 mins. in this case, pro-inflammatory cytokines such as TNF, IL-6, MCP-1 may be released in large quantities.
- Use micropipette and uptake the supernatent. Dispense into sterile centrifuge tubes. Spin gently to remove any cell debris.
- A super easy way to quantify cytokine levels is to use ELISA method. many kits are commercially available
If you don't have access to an ELISA kit, western blot is possible but it can be a little difficult due to the concentration of FBS on media and also the low quantity of the cytokine.
You would need to precipitate your culture supernatant. You could use TCA/DOC method. If you don't plan on stimulate your cells for a long time you could try serum free media or the minimal amount possible.
When I did this kind of experiment I used 2mL of FBS free medium and my control showed no bands. So it worked fine. Just be sure that your antibody is suitable for western blot and that it recognizes the active form of your cytokine.
https://www.researchgate.net/post/Is_any_one_using_flow_cytometry_for_inflamatory_factors
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