Measurement of nitrite (NO2) using Griess reagent - (Jul/25/2005 )
Am wondering if anyone with prior experience on measuing nitrite using Griess reagent kindly tell me whether this assay will be influenced by substance contained in general media (e.g. RPMI, DMEM, ...etc.).
I've been trying to follow a protocol mentioned by one article to measure nitrite from LPS-activated RAW264.7 cells. However, I always got bizzard readings when blank media containing known concentration of NO (as standards) or supernatant from cells (w/o LPS present) in that the readings (done in triplets) were jumpy quite a bit. Whereas, when plain water is used to perform the same experiment, the triplet readings were more consistent all the way through. Many thanks in advance!!
I don't think reagents in the media have major effect of griess reaction or OD that you obseve, only you shoul use media as blank
Thanks for your suggestion. And, yes, we just found out different media (DMEM w/ or w/o FBS, PBS, dH2O,...etc.) didn't have dramatic effects on the readings. The main problem in our previous assays were that bubbles introduced during the pipetting/diluting process were actually problematic. After carefully removing all bubbles in the assay wells, we now got very unified readings across all wells.