After the stimulation of macrophages with 2 drugs, I will collect cell culture medium and detect TNFa protein level with ELISA assay. What should be the best for ELISA value correction?
Another question: if to detect total TNFa protein (from the medium and intracellular), how to correct ELISA value?
Total protein? Total genomic DNA? Or some a housekeeping protein signal from western?
well i do not know your experiment design but if you have the same amount of cells in the cell culture and you measured the amount of TNFa you can present it as pg/ml if you want to correct it you have to measure the total protein in the superntant and correct it accordingly (pg/mg_protein)
I agree with Hani,
You should make enough repeats of your experiment (as long as there is room to test all in one plate) and compare to untreated cells.There is no correction needed since in the end you use mean values.
I would definitely do things in minimally of triplicate. I am assuming you are comparing to no treatment. If the drugs affect the growth of the cells, you can do relative to total protein for the lysates. But in the media, if you have serum, the total protein is not changing. So I would count the cells, and do relative to total cell number.
@Ehud, I think I must use something to correct TNFa value. Because I don't know the effects of drugs on cell proliferation and on TNFa, also we don't know the connection/relation between cell proliferation and TNFa production.
@Marcia, Thank you for the advice. I prefer to use total genomic DNA or total protein to correct, and I saw some people used Actin. I don't know which one is better.
I don't think counting cell number is good enough because of big variations.
Yes, I agree. Cell counting CNS be a problem. You can also use the glo,assay from promega which is more quantitative for cell number. but total genomic DNA might work, but there is lots of preparation for this?
Hi Xianfeng,
Sorry for my late response!
I am not absolutely sure you are right, keep in mind macrophage which produce TNF also uptake it, so basically you get somewhere along your time line an elevation in secreted protein which would diminish after a while.
Cell proliferation is very interesting to your assay, but I would not want to normalize results by is since... well, what would it mean really?
If would use them as two separate correlating (or not) data sets.
IF you want easy test of proliferation check out MTT.
Lots of kits to buy, we used Cell titer blue.
Good luck,
Ehud
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