To evaluate cell toxicity, the EZ-Cytox Cell Viability Assay Kit from DoGenBio (Seoul, Korea) was used. Cells were cultured in 96-well plates; then, five different concentrations (1, 5, 10, 25, and 50 μg/mL) of I. indigotica leaf extract in DMSO were added. The absorbance was measured at 450 nm [12 (link)].
To determine the amount of nitrite produced by macrophages, RAW 264.7 cells (1 × 105 cells/mL) were cultured in a 96-well plate for 2 h. An aliquot of 200 μg/mL I. indigotica leaf extract and dextran (10 μM) were added. Then, the cells were incubated with lipopolysaccharide (LPS) (1 μg/mL) at 37 °C for 18 h. The positive control was 500 μg/mL of aspirin. The mixture of Griess reagent and cell culture supernatants was measured at 550 nm [13 (link)].
To determine the amount of nitrite produced by macrophages, RAW 264.7 cells (1 × 105 cells/mL) were cultured in a 96-well plate for 2 h. An aliquot of 200 μg/mL I. indigotica leaf extract and dextran (10 μM) were added. Then, the cells were incubated with lipopolysaccharide (LPS) (1 μg/mL) at 37 °C for 18 h. The positive control was 500 μg/mL of aspirin. The mixture of Griess reagent and cell culture supernatants was measured at 550 nm [13 (link)].
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