RAW 264.7 cells were thawed and subcultured in 10% FBS and 1% antibiotic (penicillin and streptomycin)-supplemented DMEM culture medium. Cultured cells were kept in an incubator in a humidified atmosphere at 37°C and 5% CO2. For the cell viability assay, subcultured stock cells at around 90% confluency were collected and seeded into a 98-well plate with a cell density of 2.5×104 cells per well in DMEM supplemented with 10% FBS. Cells were left overnight and then treated with different concentration of extracts in DMEM supplemented with 10% FBS. Eighteen hours after treatment, cell viability was measured using the MTT assay kit following the manufacturer’s protocol.
LPS-induced nitric oxide (NO) production in RAW 264.7 cells is a highly exploited method in research, as a cellular inflammatory model. NO produced in this way can be quantified in a cell culture medium in the form of nitrite (NO2-), a stable degradation product of NO. For anti-inflammatory activity, the nitrite concentration in the medium was quantified by Griess reagent using the modified methods explained by Alhallaf and Perkins.43 (link) In brief, the subcultured cells were seeded at a cell density of 15×104 cells per well in a 48-well plate in DMEM supplemented with 10% FBS, and incubated overnight. Then, treatment was carried out at a non-toxic concentration in phenol red-free DMEM supplemented with 10% FBS and incubated at 37°C. One hour after incubation, inflammation was induced by adding LPS solution to make 1 µg/mL concentration in the culture medium. After 18 hours of treatment, the nitrite concentration was measured in the culture medium using Griess reagent (equal amounts of 1% sulfanilamide in 5% phosphoric acid + 0.1% naphthyl ethylenediamine di-hydrochloride in water). Then, 100 µL of culture medium was mixed with 100 µL of Griess reagent, and absorbance was measured after 10 minutes in a microplate reader at 540 nm wavelength. The quantity of nitrite in the cell supernatant was determined by comparison with the standard curve of sodium nitrite at different concentrations.
LPS-induced nitric oxide (NO) production in RAW 264.7 cells is a highly exploited method in research, as a cellular inflammatory model. NO produced in this way can be quantified in a cell culture medium in the form of nitrite (NO2-), a stable degradation product of NO. For anti-inflammatory activity, the nitrite concentration in the medium was quantified by Griess reagent using the modified methods explained by Alhallaf and Perkins.43 (link) In brief, the subcultured cells were seeded at a cell density of 15×104 cells per well in a 48-well plate in DMEM supplemented with 10% FBS, and incubated overnight. Then, treatment was carried out at a non-toxic concentration in phenol red-free DMEM supplemented with 10% FBS and incubated at 37°C. One hour after incubation, inflammation was induced by adding LPS solution to make 1 µg/mL concentration in the culture medium. After 18 hours of treatment, the nitrite concentration was measured in the culture medium using Griess reagent (equal amounts of 1% sulfanilamide in 5% phosphoric acid + 0.1% naphthyl ethylenediamine di-hydrochloride in water). Then, 100 µL of culture medium was mixed with 100 µL of Griess reagent, and absorbance was measured after 10 minutes in a microplate reader at 540 nm wavelength. The quantity of nitrite in the cell supernatant was determined by comparison with the standard curve of sodium nitrite at different concentrations.
