3.7. Interference with LPS-Induced TNF-α and IL-6 Production by RAW 264.7 Cells

The amount of TNF-α and IL-6 released by the LPS-challenged RAW 264.7 murine macrophages was measured by using mouse IL-6 ELISA MAX™ Deluxe and mouse TNF-α ELISA MAX™ Deluxe kits (BioLegend Inc., San Diego, CA, USA) [48]. RAW 264.7 cells were cultured at a concentration of 3.5 × 10⁵ cells mL⁻¹ and incubated for 24 h (37 °C, 5% CO₂). After discarding the medium, H. bhamoense trunk bark extract was added to each well at concentrations ranging from 125 to 1000 µg mL⁻¹ and incubated for 24 h. Following post-incubation, the cells were treated with LPS (1 µg mL⁻¹) for 24 h, culture supernatant being collected and stored at −80 °C until analyzed.

Protein concentration was calculated by regression analysis using a standard curve obtained with a solution of bovine serum albumin (BSA), as in Macedo et al. [48]. Colorimetric measurements were attained at 595 nm.