Hi all,
I cultured RAW 264.7 cells in DMEM high glucose medium supplemented with 10% FBS, no antibiotics, under 37oC 5%CO2.
RAW 264.7 was inoculated onto a 96-well plate at a density of 2x10^4 cells/well, incubated for 24 h under the above conditions.
Then, I stimulated RAW with LPS (1 ug/ml) after 1 hour of drug treatment. Continue incubation for 24 h and then determine the NO secretion of RAW 264.7 (by Griess assay).
However, I noticed that my cells in the edge wells on the plate secrete more NO than the rest of the wells (attached image). I have tried again and again and still don't know where the problem is.
Can you give me some advice on this?
I agree with Zhang Wehn's answer, cells seeded on 96 welll plates are very prone to evaporation. its called in my case, I put sterile PBS on the edges of the 96 well plate to prevent evaporation of wells with cells.
or fill the outer most wells with PBS close next to your seeding well. it works really good and my NO assay results triplicates were consistent. best regards
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