Measurement of Nitric oxide from LPS stimulated Raw264.7 cells?

11 August 2025 2 6K Report

HI all, I am trying to assess the antiinflammatory effect of herbal extracts by measuring the NO release in LPS stimulated Raw cells. I am seeding 200 ul of 5x 104 cells in a 96 well plate on day 0, using 10 % FBS media. After 24 hours, the media is replaced with 1 % fbs media containing variable concentrations of extracts for 1 hour before inducing inlfammation with 1 ug/mL LPS. After a further 24 hours, the amount of NO relased in the 100 uL of supernatant is measured using Griess reagent.

Please advise if the amount of NO calculated should be normalised to the % of viable cells at respective concentration point? The cell viability at it concentration point is measured using the MTT assay.

I cannot find peer reviewed articles describing the normalisation of NO release with cell number.

thank for your advise.

best regards

Zein Al-Abideen Douba

Yes, normalizing NO release to cell viability is highly recommended when using LPS-stimulated RAW264.7 cells, especially if your treatments (herbal extracts) may affect cell survival. This ensures that observed differences in NO production reflect true changes in inflammatory response rather than simply differences in cell number.

Key points:

Normalized NO=NO (µM)% viable cells\text{Normalized NO} = \frac{\text{NO (µM)}}{\text{\% viable cells}}Normalized NO=% viable cellsNO (µM)Normalization approach: Measure NO release in the supernatant using the Griess assay. Measure cell viability at the same concentration using MTT or a similar assay. Normalize NO concentration to viable cell number or % viability at that treatment point.Example:

Rationale: Treatments that reduce viability may reduce NO release simply due to fewer cells. Normalization allows accurate comparison across concentrations and avoids underestimating anti-inflammatory effects.

Supporting literature: Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., & Tannenbaum, S.R. (1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Analytical Biochemistry, 126(1), 131–138. DOI Olivares, M., Gonzalez, C., & Gonzalez, M. (2013). Normalization strategies for NO measurement in cell culture studies. Journal of Immunological Methods, 387(1-2), 47–53. DOI

Tip: Always include vehicle controls and verify that your extract solvents do not interfere with the Griess reaction

Nawraj Rummun

thank you for the detailed help. best regards

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