We have tested different concentrations of LPS Salmonella typhimurium (50-100 ng / mL up to 8.1 microg / mL) unsuccessfully. Apparently it should be simple but maybe there is some detail we did not consider.
I wonder if you might need to co-treat the cells with Interferon gamma. I have no personal experience from working with RAW 264.7 cells, but some macrophages require Interferon stimulation. How do you detect the activation?
Thanks Sandra Fernaeus measure nitrite levels (Griess reaction)
Paul Dias I had not considered that factor, we are working with cells of 25 passages approx
We read some articles and use mixtures of LPS-IFN, is the most recommended? IFN should be from mouse? amounts that you recommend?
If possible to find rabbit IFN it would be my first choice. I have not tried IFN/LPS myself, since my mice BV2 cells are activated by LPS only. Amounts of IFN used are usually very low in nano or picomolar range. You have to find what others have used and test the optimal conditions for you. But you should also consider the other advices given above. Best of luck.
everyone have good points to be analyzed. however, you usually don´t need to add IFNg. 1ug/ml is a standard conc for raw cells and should be sufficient to induce NO. how does your LPS work with other cells? do you always sonicate LPS before using it? if you don´t, then it my be the problem. best!!!
Fabianno Dutra, We have not sonicated LPS, Will this be the problem? why?
hi francisco,
sonication is important to disrupt the micellar agregates formed by LPS. This happens because LPS is very hydrophobic. Resarchers argue that micella of LPS doesn´t bind properly to cells thus increasing the concentration needed to induce activation. i suggest you to prepare a stock of 1mg/ml (maximum) and eventually sonicate it. you can store LPS in lower concentrations, ready to stimulate cells. but in this case i recommend you to use glass instead of plastic and always sonicate it before stimulation. LPS can stick to plastic and reduce the concentration of your stock.
best!
My experiences disagree with Fabianno, actually. I regularly stimulate RAW cells with E. coli O111:B4 LPS (Sigma) without needing to sonicate. I store the LPS at 5 mg/mL at -20C and dilute to 100 ng/mL final concentration for stimulation. I do, however, agree with him that IFN is unnecessary.
I've had no issues with getting RAW to produce pro-IL1b within 2 hours and also see really distinct NFkB translocation within 1 hour at concentrations as low as 10 ng/mL. I have not tested lower concentrations yet.
If you store you stock-solution of LPS in certain plastic vials, these may bind LPS. This may result in a lot lower concentration in you cell culture. We use glass vials to store our LPS stock.
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