Cells were incubated in round-bottomed 96-well microplates (Corning Incorporated, Corning, NY, USA) at concentrations of 1 × 104 L5178Y-R cells/well and 1 × 105 PBMC/well in complete RPMI 1640 medium. After 24 h of incubation, cells were treated with methanol extracts at concentrations ranging from 3.9 µg/mL to 1000 µg/mL. The antineoplastic vincristine sulfate (VC) (Hospira, Warwickshire, UK) at 100 µg/mL was used as a positive control and untreated culture medium was used as a negative control [15 (link)]. Cells were incubated for 48 h at 37 °C in an atmosphere of 5% CO2 in air and cell viability was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT; Affymetrix, Cleveland, OH, USA) colorimetric method by adding 15 µL of MTT/well (0.5 mg/mL final concentration) and incubating the plate at 37 °C for 3 h [18 (link)]. Plates were then decanted, formazan crystals were dissolved with 100 µL of DMSO, and optical densities (OD) were measured at 570 nm in a microplate reader (MULTISKAN GO; Thermo Fisher Scientific, Waltham, MA, USA). Percentage growth inhibition was calculated as follows (2):
Free full text: Click here
