*2.3. 3-(4,5-Dimethylthiazol-Zyl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay*

A7r5 cells were seeded onto each 24-well plate (Corning Inc, Corning, NY, USA) at a density of 10<sup>5</sup> cells/mL, and treated with various concentrations of TNF-α (0–20 ng/mL) alone or TNF-α (10 ng/mL) in combination with HLP (0–1.0 mg/mL) for 24 or 48 h. Thereafter, the medium was changed, MTT (0.1 mg/mL, Sigma, St. Louis, MO, USA) was added for next 4-h incubation. The viable cell number was directly proportional to the formazan production, which was solubilized in isopropanol and detected at 563 nm with a spectrophotometer. The MTT assay was used to evaluate the effect of TNF-α alone or TNF-α and/or HLP on cell viability, and to determine the non-cytotoxic doses of HLP, as described by Chen et al. [16].
