MTT assay and lactate dehydrogenase (LDH) assay

AMC-HN-8 cells (5×10⁶ cells/well) were plated into a 96-well plate for 24 h at 37°C. The cultured cells were treated with various concentrations of Ophiopogonin D (0, 12.5, 25 and 50 µmol/l) for 12 or 24 h. Subsequently, 20 µl MTT (5 mg/ml) was added daily to each well and incubated for an additional 4 h at 37°C. Dimethyl sulfoxide (200 µl) was added to each well and dissolved for 10 min, and the absorbance at 490 nm was measured using a plate reader (Synergy-HT; BioTek Instruments, Inc., Winooski, VT, USA).