I need to test the stimulation production of TNF alpha in the RAW cell line by LPS. I am looking for an easy and cheap experiment to quantify the serial dilution stimulation in this macrophage cell line.
I guess you have probably already considered ELISA, which would be the standard way of doing this. It's easy. With about $5 / test in 96-well format, I would also call it reasonably priced, at least in comparison to what it costs to do cell culture at all.
If you must bring the cost per test down, and have many tests, then you could use a bioactivity assay with a reporter cell line. Buying such a cell line for the first time costs top dollar, but after that you can propagate it on your own. For example, many vendors sell NFkB-luciferase constructs that would work great with TNFa.
Another option might be to develop your own antibodies and compose you own ELISA kit. That's a truly epic upfront investment, but it will be nearly free ever after.
ELISA, as suggested by John is one possibility. If you are searching for an easy way for a (semi) quantitative measurement of your cytokines, I like to use a intracellular staining and flow cytometry. If you are interested in this, I can share my protocol with you. We usually use whole blood and measure the cytokine expression (TNF, IL1b, IL6, INFy IL17) in monocytes and lymphocytes.
Be aware of the necessarity of using brefeldin A or monensin so you are able to measure the cytokines within the cells.
Best regards
Johannes
Thanks, Johannes Zeller. Could you share with me the protocol for intracellular staining and flow cytometry? I will have another assay and that would be very useful.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line