Cell Proliferation Assay (MTT Assay)

HT-29 cells were cultured in 96-well plates at the density of 1 × 10⁴ cells/0.1 mL/well. Cells were serum-starved for 24 hours and then incubated with a series of concentrations (0.157-2.5 v/v %) of TXL and its constituent fractions (EA, BU, and WA) in triplicates for an additional 24 hours at 37°C in a humidified 5% CO₂ incubator. Mouse splenocytes were cultured at 4 × 10⁵ cells/0.1 mL/well in 96-well culture plates in the presence of a series of concentrations of TXL and its constituent fractions in triplicates for 48 hours at 37°C in a humidified 5% CO₂ incubator. The procedure of cell proliferation was carried out in accordance with recommendations of the manufacturer (Cell Proliferation Kit I, Roche Applied Science, Madison, WI, USA). All reported values are the means of triplicate samples. IC₅₀ (half-maximal inhibitory concentration) was calculated by SigmaStat Statistical Analysis Software.