I just started my PhD in pharmacology department and currently i'm working with 264 macrophages cells.My problem is these cells were not activated by lps.the absorbance are same as the unstimulated cells in the griess assay.I seeded the cells in 96 well plate 60000 cells in 0.1mL in each well using 10% FBS,1% antibiotics and 1% Glutamin.then after 24 hour incubation added 1000,500,250,125,62.50 ng/mL LPS along and the same concentrations of LPS with constant concentration of IFN (1000ng/mL).Then measure the nitric oxide concentration by griess assay after 24 hour incubation period.I have done this in several time but the problem is these cells were not activated.Did anyone has the same problem and resolved this problem?
Dhanu
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