First, the Griess assay measures nitrite (and nitrate, if you use a reductase to convert it to nitrite) and not nitric oxide. Nitrite is an aqueous oxidation product of NO, and can be considered to be an indicator of NO bioavailability. If NO is being produced, then over time nitrite should accumulate in your media. So you want to sample media and use the Griess assay to measure the nitrite content in the media.

The longer you allow the cells to make the NO and thus the nitrite to accumulate in the media, the more likely you are to detect something. For example, if you don't detect any nitrite after 72 hours of culture, then you likley won't after 48 either. When you change the media, you reset the concentration&clock back to zero. Remember the volume of the media above the cells will determine the concentration, so try to keep the volume low, no more than 100 uL for a 96 well plate and be aware of possible evaporation related edge effects.

Finally, make sure you run your controls and calibration standard curve in the same media!

Peter is right. Basically culture your cells, collect the cell supernatants and use those in your Griess Assay. I have used this kit from Thermo-Fisher and they have a protocol that could help.

https://www.thermofisher.com/order/catalog/product/G7921

Hi,

Few things to consider in your NO assay:

- Make sure that when culturing in 96-well plate, your sample size is enough to process during the assay/prepare multiple wells per condition.

- The Greiss reaction can be done in an enzymatic or non-enzymatic assay. If you opt for the enzymatic assay, make sure that there is nothing in your supernatant (mainly your treatment) that interfers with the enzyme activity.

This is the kit I used: https://www.oxfordbiomed.com/sites/default/files/spec_sheet/NB88.121218.pdf