3.15. Nitric Oxide (NO) Production in RAW264.7 Macrophages

The RAW 264.7 macrophages were cultured in The RPMI 1640 medium (Hyclone, Logan, UT, USA) containing 10% FBS. The compounds were dissolved in DMSO and further diluted in medium to produce different concentrations. The cell mixture and culture medium were dispensed into 96-well plates (2 × 105 cells/well) and maintained at 37 °C under 5% CO2. After preincubation for 24 h, serial dilutions of the test compounds were added into the cells, up to the maximum concentration 25 μM, then added with LPS to a concentration 1 μg/mL and continued to incubate for 18 h. The amount of NO was assessed by determined the nitrite concentration in the cultured RAW264.7 macrophage supernatants with Griess reagent. Aliqueots of supernatants (100 μL) were incubated, in sequence, with 50 μL 1% sulphanilamide and 50 μL 1% naphthylethylenediamine in 2.5% phosphoric acid solution. The sample absorbance was measured at 570 nm by a 2104 Envision Multilabel Plate Reader (PerkinElmer, Inc., Waltham, MA, USA). Dexamethasone was used as a positive control.