Hi everybody,
I am working with a RAW 264.7 cell line to test the mRNA expression of the proinflammatory cytokines. Right now, I'm having some problems, hoping to get some useful advice.
RAW 264.7 cell line was cultured in RPMI medium supplemented with 10% FBS (no antibiotics). Culture conditions 37oC and 5% CO2.
I inoculated with a density of 8x10 ^ 5 cells / well ( 6-well plate). After 24 hours of incubation with the above conditions, I treated the cells with 1 ug/ml concentration of LPS. After 24 hours, the culture medium was measured for the NO value (Griess assay, NaNO2 calibration curve); however, the NO value was very low (below 5 uM) (no difference between the LPS-treated sample and the control)
Observed under the microscope, the RAW cell morphology (cultured after 24 hours) (as shown in the picture) was like the cells under stress.
I think that this cell line has been altered after many passes, so LPS does not inflame it.
Dear Duy Huynh,
you can check intracellular iNOS activity by immune fluorescent or immune chemistry on cell culture after LPS application.
Good luck
Dear Duy,
I believe you are facing a cell density problem. I usually do stimulation on Raw cells with LPS and its works fine. Try something around 2-3 X105 cell in a 96 well plate (100 ul culture) for 24 hours. LPS purity its important to get a good response. I use a range from 100 to 500 ng/ml depending how pure is LPS and its works fine. Maybe you are pushing too much LPS and getting your cells killed.
Good luck
You can add ATP as second signal to activate your cells after LPS treatment. You can keep one positive control with IFN-g for comparing the results with your test. I am facing the same problem however with bacterial infection and not with LPS, but I am getting desired results with IFN-g.
If your cells have been passaged too many times or if they are infected with Mycoplasma, they may not respond to TLR -- and hence LPS -- stimulation. You may also want to try a different batch of LPS.
Check out the following:
Article Mycoplasma Suppression of THP-1 Cell TLR Responses Is Correc...
Hi, I used cDMEM high glucose media supplemented with 10% FBS and 1% penicillin/ streptomycin.
and try seeding your RAW 264.7 in 96well with higher cell density.
incubate till, 50~60% confluent. then treat it with your LPS 1ug/mL then incubate for 24hrs.
collect sup then proceed to NO assay. it works really fine.
Dear Duy Huynh.
Hope your problem was resolved by this time. Still, if you are facing a problem with the experiment, use DMEM complete media that is good for culturing adherent cell lines and seeding the cells just two hours before starting the experiment, and don't go for the usage of higher LPS concentration. It will work
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