Quantification of nitrate/nitrite levels and the cell death induced by different nitric oxide (NO) donor compounds. Chondrocytes were obtained from the femoral condyle and the tibial plateau of knee osteoarthritis patients, cultured in monolayers, and seeded at 4 × 104/100 μl/well in 96-well microtiter plates. First-passage chondrocytes were used in the subsequent experiments. Chondrocytes were treated with the respective NO donor compounds for 24 hours. (a) Chondrocyte culture media were harvested and analyzed with a nitrate/nitrite colorimetric assay kit, as described in Materials and methods. (b) Cell death was quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide (MTT) assay. MTT was added to each well after the collecting medium. Percentage cell survival was calculated by dividing the optical density of treated cells by the optical density of untreated control cells, and multiplying by 100. Cell survival in control culture was set at 100%. * P < 0.05 versus control. Data shown are the means and standard deviations of duplicate experiments on three different donors. NOC-5, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetyl-L-penicillamine.
Kim et al. Arthritis Research & Therapy 2005 7:R526-R535 doi:10.1186/ar1705