A microplate reader

A549 cells were counted in logarithmic phase and 6000 cells (90 μL volume) were placed in 96-well plates. 10 μL varying concentrations of Tan IIA (final concentrations 80, 60, 40, 30, 20, 15, 10, 5 and 2.5 μmol/L) and ADM (final concentrations 8, 4, 2, 1, 0.5 and 0.25 μmol/L) were added into drug groups, while negative control group (vehicle group) was only added 10 µL DMSO or normal saline without Tan IIA or ADM. Cells were incubated for an additional 2 h with CCK-8 reagent (100 μL/mL medium) and the absorbance was read at 450 nm using a microplate reader (BioTek, Winooski, 126 VT, USA). Cell proliferation inhibition rates were calculated according to the following formula: the proliferation inhibition ratio (%)=1−[(A1−A4)/(A2−A3)]×100, where, A1 is the OD value of drug experimental group, A2 is the OD value of blank control group, A3 is the OD value of the RPMI1640 medium without cells, and A4 is the OD value of drugs with the same concentration as A1 but without cells. The IC₅₀ (50% inhibitory concentration) value, which represents the concentration of the drug that demonstrates 50% of cell growth inhibition, was calculated by nonlinear regression analysis using GraphPad Prism software (San Diego, USA).

After treatment with siRNA for 24 h, the cells were reseeded in 96-well culture plates at a density of 5,000 cells/well and incubated for another 48 h. Then, 10 µL CCK-8 solution was added to each well and incubated for another 2 h at 37 °C. The absorbance was analyzed at 450 nm by utilizing a microplate reader (BioTek).