RAW cells are a pain but a useful tool. When grown for too long, or in media with even slight levels of endotoxin (LPS) their TLR pathway becomes tolerant/desensitized. When you come along later with a proper LPS challenge (50-100ng/ml) for 8-24 hours you get nearly no response. Standard readout, TNFa ELISA.
Improving your cell culture is a good place to start. Confirm no mycoplasma. After this try growing cells in media + FCS from different vendors. Don't culture your cells too long and try to develop an approach where the most fresh healthy cells give a quick and decent TNFa response in an overnight LPS stimulation. This may take some weeks or even a month or two of part time work. Once you have proper cells expand them quickly under the same conditions and freeze down many tubes.
Also use good quality LPS from InVivogen or another Ultra-pure source. LPS from classic vendors like Sigma may, or may not, have agonists for TLRs or NLRs. In the end, if things don't improve get RAW cells from a trusted cell source like ATCC and then culture what you thaw as above so that you don't "desensitize" the newly arrived and expensive cells.
Good luck!
Thank you very much for your response. It is so helpful.
I've tried challenging with 1µg/mL and 10µg/mL of LPS (Sigma) for 24 hours (1 millions cells in 25cm3 flask) but it didn't work. Maybe I should try LPS from other vendors.
Your dose is very high. If nothing happens with this your cells are desensitized and you need new ones. I would think your LPS is fine.
But my cells are actually new ones from ATCC (TIB-71). I started using it after passage 5 and it didn't work.
How did you passage your RAW cells? by using trypsin? Trypsin is not good to RAW cells. And how much is about the NO base of your RAW cells? THe normal RAW cells' NO base is about 2um.
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