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SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 2172.6. Evaluation of in vitro anti-inflammatory activityRAW 264.7 cell line was cultured in Dulbecco%u2019s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Cells of 2%u00d7105 cells/well were prepared and incubated for 24 h at 37%u00b0C and 5% CO2. DMEM medium without FBS was used as a replacement and cultured for 3 h. Different concentrations of tested samples were added and incubated for 2 h before cells were stimulated to produce NO with 10 %u03bcg/mL lipopolysaccharides (LPS) for 24 h.The reaction to determine the presence of NO consisted of 100 %u03bcL of experimental cell culture sample and 100 %u03bcL of Griess reagent, incubated for 10 min at room temperature. The absorbance was measured at 540 nm [9]. The reference material was NG-Methyl-L-Arginine acetate (L-NMMA) (Sigma), the negative control was the cell culture medium, and the positive control was the LPS-stimulated cell sample. Sodium nitrite (NaNO2) standard curve was used to determine NO2%u00af content.The MTT method was used to determine the cytotoxicity of the experimental extracts against RAW 264.7. The cell staining reaction with MTT was performed by adding 10 %u00b5L MTT (5 mg/mL) for 4 hours. The medium was removed before the formazan crystals were dissolved with 100 %u00b5L DMSO. The absorbance at 540 nm was determined. The negative control sample is the cell culture medium, the positive control sample is the cell sample without the experimental extract added.