4.2. RAW 264.7 Cell Culture and Cell Viability Assay

RAW 264.7 cells, a murine macrophage cell line, were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) [with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (PS; Hyclone, Logan, UT, USA)] in a humidified atmosphere containing 5% CO₂ at 37 °C. Cells were stimulated by replacing the culture medium with medium containing 1 μg/mL LPS (Escherichia coli 011:B4, Sigma Chemical Co., St. Louis, MO, USA), and incubated with P. deltoides leaf extract for 24 h.

Cell viability was assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 4 × 10⁴ RAW 264.7 cells per well of a 96-well plate were incubated with various concentrations of P. deltoides leaf extract (0–200 μg/mL) at 37 °C for 24 h and then the medium was completely removed. The cells were washed and treated with 50 μL of MTT, after which the plates were incubated at 37 °C in the dark for 3 h. The resultant formazan crystals were dissolved in 50 μL of dimethyl sulfoxide, and the absorbance was measured at 570 nm using an ELISA reader (Tecan, Männedorf, Switzerland).