I am trying to peform Griess assay using the cell culture superntant. I used DMEM (High Glucose) +1%antibiotics and 0.5%FBS for culturing the RAW264.7 cell lines. Then I treated the cells with the synthesized peptides. After tretment, When I tried to performed the griess assay using the invitrogen kit, immediately after adding the cell culture supernatant into griess reagents, it turned yellow. Is it because of the phenol red in culture media or any other factor?
Dear Priti Panchal According to Wikipedia a typical commercial Griess reagent contains 0.2% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2% sulfanilamide in 5% phosphoric acid.
The appearance of Griess reagent could be due to presence of FC reagent to the compound, the reaction between 0.2% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2% sulfanilamide could be reason for addition of this molecule.
The immediate yellow colour you observed after adding the cell culture supernatant to the Griess reagents is most likely due to the presence of phenol red in your DMEM culture media. Phenol red, a pH indicator included in many culture media formulations, can interfere with colorimetric assays like the Griess assay by contributing to the background color or altering the expected color change.
If phenol red is not the cause, consider other components in the media or treatment that might be affecting the assay. Synthesized peptides or their interactions with Griess reagents could also potentially cause color changes. Perform a control experiment using an untreated culture medium and Griess reagents.
Hello Priti Panchal
Nitrite is determined through formation of a reddish purple azo dye produced at pH 2.0 to 2.5 by coupling diazotized sulfanilamide with N-(1-naphthyl)-ethylenediamine dihydrochloride (NED dihydrochloride). In other words, the sample containing nitrite is reacted with Sulfanilimide and N-(1-naphthyl)-ethylenediamine to form a colored species that absorbs at 540 nm.
This Griess Reagent System is based on a chemical reaction that uses sulfanilamide and N-1-naphthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. So, please check the pH.
DMEM (high glucose) is buffered for high CO2, and sodium bicarbonate in DMEM can cause the pH to become basic if CO2 is removed.
Phenol red does not interfere with Griess reaction. It will interfere with the absorbance reading taken at 540nm. It is therefore recommended to use media without phenol red to avoid any interference in the absorbance readings.
Best.
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