I am using Modified Griess reagent for measurement of NO production form astrocyte like cell line, RT-PCR results showed strong response from the cells to cytokines treatment by expression of iNOS. I tried to measure NO using cells grown on DMEM medium with 2 % fbs after 12, 24 and even 48 hours after cytokines treatment but griess shows no color change. I made the standard solutions from sodium nitrite from 1 um to 80 um using the same medium , and the standard solutions color had no problems.
I am using 6 cm culture dishes for my experiments , I talk the medium supernatant then centrifuge for 6000 rpm at 5 min , then I talk 100 ul from medium supernatant and same amount from griess reagent. I incubated at room temp for 15 mins , but with no change in color. Only the standard solutions give colors.
Do you have any idea about this problem?