Without any activation, I constantly observe activated RAW 264.7 cells in culture confirmed through cytokine assay of TNF. Even after a number of passages (Passage number 25 to 40) are given, TNF remain high. Finally, I switched to a different stock of RAW 264.7 cells but still the problem persists. I suspect probably the autoclaved distilled water used to dilute 10X PBS to 1X PBS may contain low amounts of endotoxins which are leading to activation state of RAW 264.7 cells?
Can anyone explain why RAW 264.7 cells are constantly in activated state? Can autoclaved distilled water contain enough endotoxin levels to activate RAW 264.7 cells? And how to circumvent this problem?
Dear Sanjai Autoclaving does not remove/inactivate endotoxins. Best to use water checked for the presence of pyrogens (sold as Pyrogen-free water). Check the water for LPS using a LAL assay. Most big pharmaceutical companies and blood transfusion labs do these assays on a routine basis and will also have commercially available Pyrogen-free water.
Dear Sanjal,
endotoxin still acts at extremely low concentrations. A notorious source for LPS contaminations are, for instance, glass surfaces. Working with LPS-sensitive cell lines you need to make sure you ONLY use plasticware (no glass pipets for instance) and all reagents (FCS) should be low endotoxin-certified. Even using glass bottles for the 10x PBS might be a reason. It sounds crazy , but from past experience (Mono Mac 6 cells) I can confirm its true. you may need to consider buying custom-made PBS even. Iif you then keep the cells under LPS-free conditions for several passages, they may calm down again and become usable again. For some data and background on this have a look at our old paper: http://www.jbc.org/content/269/25/17001.full.pdf
Good luck!
Thomas :-)
After giving repeated passages to RAW 264.7 cells, can they return to normal resting state?
Sanjal, I would expect they can. However, I have only worked with Mono Mac 6 . You will have to test that before you can do your experiments: Just keep measuring your TNF-levels in the medium for 4-5 passages to see if it heads down. every now and then, you could throw in a small stimulation with LPS in a little flask or multi well-format to see the level of activation and you can see when you reach the -fold induction published in the papers. Its tedious, but necessary, In am afraid. Once its up and running, you should get good data. Are you tied to Raw cells, or could you change cell line, worst case?
Again, good luck!
Thomas :-)
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