I used RAW264.7 (2 x 106 cells in 100 ul) at below passage 12 and induced with LPS at concentration of 0.1, 10 and 100 ug/ml and incubate for 22 hours. I measure the nitric oxide level and found that there is no difference of nitric oxide level between control (0 ug/ml LPS) and LPS-induced RAW cells as well as between different concentration of LPS. How to solve this problem?
Hello, firstly, your seeding density may be too high. I recommend a density between 2x10^5 cells/mL and 4x10^5 cells/mL (100 uL per well). After seeding your cells, allow a recovery period of about 14-16 h, but not much longer (or the cells will start to proliferate and alter your expected density).
0.1 ug/mL LPS is already considered quite a potent dose. LPS doses beyond 1 ug/mL are not recommended as they are considered more or less supraphysiological, and do not mimic things realistically.
Lastly, check if your cells (before LPS addition) are in a happy resting state. Sometimes cells get activated by microbial contamination or simply overly high density which expedites cell death.
Reduce your FBS concentration to 3%, instead of 10% for LPS treatment.
Perhaps you can add a bit of Interferon gamma (100U/mL) together with the LPS
hello, please check Macrophage cell for other contamination, and check LPS activity. perhaps LPS loss its structure.
I subscribe to all the points above mentioned. I want to add that cell line passage is also and important factor because if you cross a specific limit of the passage cells get activated phenotype and you don,t get your desired results.
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