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I need some help to differentiate my U937 cell line with PMA, stimulate with LPS and produce nitric oxide. I tried different concentrations of pma, lps and medium and there was no NO. I need a real good protocol in order to start my experiment. So far i've managed to differentiate the cells but no NO. I seeded and stimulated 700000 cell/1400µl/well in 6 well plates in RPMI medium without FCS, with 10, 100, 200 nM PMA and I saw macrophagues. I took out the supernatant and stimulated for 24 h with 1 and 10 µl LPS in 500 µL normal medium. After 24 h I proceeded the Griess reaction using 150 µL supernatant and the same volume of Griess reagent and I had no nitric oxide.
Some papers use a reductase for the Griess reaction, but others don't. Should I buy a reductase?
What do I do wrong? Please, can you help me?
Thank you
Petruta G.
