Collection of peritoneal macrophages and nitrite assay

Male Balb/C mice were i.p. injected 4% sodium thioglycollate media 1 mL per mice. Under aseptic conditions mice were euthanized after 24 h and injected 10 mL of RPMI 1640 medium in peritoneal cavity. After 5 min, the medium was taken out and centrifuged at 1800× g for 10 min at 4 °C. The cell pellet was resuspended in RPMI 1640 medium. The macrophages (3× 10⁶) were cultured in 96 well micro-plate in the presence of LPS (1 μg/mL), β-Methasone (0.001 μg/mL) and variable doses of methanol extract (25–200 μg/mL). The plates were incubated at 37 °C in a humid saturated atmosphere containing 5% CO₂ for 24 h. Supernatants were collected after centrifugation and stored at −80 °C for determination of cytokines. The supernatants were used for nitrite assay using Griess reagent. For the NO₂ assay (nitrite content), 100 μl of the culture media was incubated with 100 μL Griess reagent (0.1% naphthyl-ethylenediamide and 1% sulphanilamide in 2.5% phosphoric acid solution) at room temperature for 10 min in 96 well micro-plate [16]. Optical density was measured at 540 nm using ELISA plate reader. The nitrite concentration was determined by extrapolation from a sodium nitrite (NaNO₂) standard curve and the results expressed in μM.