Hello all,

I've been trying to perform a Nitrite/ Griess assay on isolated cell media but no detectable traces of Nitrite is present.

The media itself is phenol red-free MEM supplemented with 10%HIFC, 1% Pen/strep, 1% NEAA and 2mM L-glutamine; the media is further supplemented with various reagents and was incubated at 37C for 24hrs with HeLa cells. The reagent concentrations were within a range known to be on either side of the IC50 value for the cell line in use.

For the assay, sodium Nitrite was used as the standard and produced colour each time  the Griess reagent was added. (absorbance values correlate with concentration used, 0-35uM). Media was placed into the wells undiluted and demonstrated no colour development.

The Griess reagent used is composed of 0.5mg/mL of NED, 5mg/mL sulfanilic acid and 2.5M phosphoric acid.

After the addition of the reagent, the standard curve samples develop quickly, usually within 30 seconds, though the plate is covered for 15 minutes. 

At this current stage of this assay, detection of nitrite and not nitrate is the primary focus, hence the addition of reductase and co-factors have not been Incorporated.

Various methods have already been utilised to try address the issue and achieve a distinct reading. Originally MEM with phenol red was used but soon omitted due to the varied readings received. Plate incubation time was also increased, 1 hr, to no avail.

Any help regarding this would be appreciated.