I treated RAW cells by LPS at passage 5 and 6 for 24 hr. I used DMEM (10%FBS) with penstrep. Griess reagent was used to measure NO release. No repsonse after LPS stimulation for 24 hr. Any help?
There are two things you try, 1) try lowering the volume of media in the wells (i.e. have a high cell to media volume ratio), this will help concentrate secondary signals and also the NO once it is made so you can detect the nitrite by Greiss assay easier. 2) add interferon gamma at the same time as LPS, a lot of the time NO will not be produced due to high arginase activity, adding interferon gamma usually shuts down arginase (LPS actually increases it), this is one of the reasons why traditional 'M1' polarization requires interferon gamma.
Hi Taddesse,
One suggestion would be to count viability of your cells after stimulation. You may have dead cells and that is why you are not seen any response to you stimulus. Raw can be intact but in an final apoptotic stage, so you would see perfect cells under the microscope, but your cells wouldn't be viable.
You should start a new freshly grown lot from lower passage. If RAW264 cells do not respond to LPS after 24 hrs it is unlikely to respond after 48hrs. These cells tend to unexpectedly shut down their nitric oxide production even when it keep great morphology and high viability. Do not waist time on this lot start fresh.
There are two things you try, 1) try lowering the volume of media in the wells (i.e. have a high cell to media volume ratio), this will help concentrate secondary signals and also the NO once it is made so you can detect the nitrite by Greiss assay easier. 2) add interferon gamma at the same time as LPS, a lot of the time NO will not be produced due to high arginase activity, adding interferon gamma usually shuts down arginase (LPS actually increases it), this is one of the reasons why traditional 'M1' polarization requires interferon gamma.
Hello Taddesse
I think that you can do differents actions for resolve this problem.
1) Check whether this problem come from same aliquot and this is reproducible. Perhaps, there will be a mycoplasma contamination.
2) Check the cell survival or viability before your LPS treatment.
3) You can try test to different LPS concentration to find a good condition. May be can change your source of LPS (other commercial reagent or diferent type of purifiation).
4) Test some molecules known to LPS activation by PCR, as functional control. For example, miR-146a or NFKB. In addition, eNOS.
Whether after all this is Ok. you should test other system to detect NO.
Regards
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