Softmax pro m5e

The hiPSC-CMs plated in the 96-well white assay plate were exposed to various concentrations of the five test compounds. After 2-day exposure, total intracellular adenosine triphosphate (ATP) level was measured using ATPlite Luminescence ATP Detection Assay System kit (PerkinElmer, Netherlands), according to the manufacturer’s instructions. Briefly, viable cells were lysed by incubation with a mammalian cell lysis solution for 5 min on an orbital shaker. After cell lysis, the substrate solution was immediately added to all wells, and the assay plate was shaken for another 5 min. Following a 10 min incubation in the dark, luminescence levels were measured on a Softmax Pro M5e 96-well plate reader (Molecular Devices). Luminescence values are directly proportional to the amount of ATP.

Cell cytotoxicity/membrane damage induced by ETP was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. The iCell-CMs were seeded on a fibronectin-coated (5 µg/cm², 2 h at 37 °C) 96-well plate at a cell density of 20 × 10³ cells per well in quadruplets. Cells then exposed to 10, 15, and 30 µM of ETP for 48 h. Following the exposure, the culture medium was aspirated and centrifuged at 3000 rpm for 5 min to obtain a cell free supernatant. The activity of LDH released into the medium was estimated using Pierce™ LDH Cytotoxicity Assay Kit (Thermo Scientific™) according to the manufacturer’s instructions. The absorbance from formazan at 490 nm was recorded using a Softmax Pro M5e 96-well plate reader (Molecular Devices, Sunnyvale, CA, USA). The raw data were analyzed and presented as percent cytotoxicity with respect to that of control with ± SD.