We recently purchased macrophage like cell RAW 264.7 from European collection of cell cultures. The cells have been thawed according to original handling procedure for frozen cells. We have now passage no.10. They seems completely healthy, no sign of contamination, no visible morphological changes. Unfortunately, the cells do not respond to LPS (10-500 ng/mL) at all, measured with Griess reagent as NO production. Griess reagent is new and fresh. The positive control (LPS) does not differ from negative control +/- 4 uM NO. LPS was previously tested on "old" RAW cells and it worked even at low concentration 10 ng LPS/mL.
Because these cells are new and I want to avoid to buy new one, I kindly ask you for some suggestion what can I do? Should I try to apply CSF1 (M-CSF) or any other stimulus to stimulate them to start respond? Or does exist some other easier solution?
Not all LPSs are equal - I'd try several - Ecoli and Salmonela ought to induce a response. Then you may want to try more traditional response markers such as IL-1, IL-6, TNF and IL-10. Personally I think you did the correct thing in acquiring the cells from a "primary" source rather than a lab where, as the previous respondent said, the activity may have 'drifted".
If the "old" cells were working, why did you change?
Good luck!
G
There are two sublines of Raw264.7 cells both available from ATCC. The one with cat no CRL-2278 is (NO-). It will not generate NO with a single stimulus LPS or IFNg but it does if you combine them. The one with TIB-71 cat no works just fine with LPS
Raw are notoriously tricky to handle for some obscure reason.
We are using them in InvivoGen for multiple mTLR4 stimulation and I can confirm that this pathway is active on the nf-kb side (IL6, TNF or reporter). If I was confronted to your trouble, I would also have a look into my LPS content in serum (largely overlooked by most labs but real trouble) or mycoplasma contamination. You may have tolerised your macrophage...
Pretreatment with IFN-g or serum starvation or both should potentiate your RAW cells.
Hello Ondrej
iF you still have the old RAW ,you can try activate your new one by adiing 1:1 old/new medium (old medium = supernatant mediium from the old growing RAW). If this did not work try to purchase from ATCC.,Good luck.
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