Varioskan flash 2.4 microplate reader

SK-BR-3, HCC1954, and KPL-4 cells were seeded into 96-well plates at a density of 3,000 cells/well. The next day, the cells were treated with T-DM1 (0, 0.01, and 0.03 μg/mL) in the presence of the test compounds. Then, 4 days later, cell viability was determined using Cell Counting kit-8 (Dojindo). Absorbance at 450 nm was measured with a Varioskan Flash 2.4 microplate reader (Thermo Fisher Scientific). Cell viability was calculated as a percentage of untreated cells.

The 661 W cells were seeded at a density of 3 × 10³ cells per well in 96-well plates, and then incubated for 24 h under a humidified atmosphere of 5% CO₂ at 37°C. After the addition of NAC 1 mM or vehicle (1% FBS, DMEM), the cells were incubated for 1 h and then exposed to 0.38 mW/cm² or 2,500 lux of blue, white, or green LED light for 6 h or 24 h. Then 10 μM of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; Invitrogen, Carlsbad, CA, USA), a free radical probe, was added to the cell culture after LED light exposure and incubation was continued for 1 h at 37°C. The radical probe was converted to 2′,7′-dichlorodihydrofluorescein (DCFH) by the action of intracellular esterase. Intracellular DCFH (nonfluorescent) was oxidized to 2′, 7′-dichlorfluorescein (DCF, fluorescent) by intracellular ROS. Fluorescence was measured by a Varioskan Flash 2.4 microplate reader (Thermo Fisher Scientific) at 485 nm (excitation) and 535 nm (emission).