Spectramax3 microplate reader

A549 cells pretreated with W-BR, E-BR, SSa, and SSd for 24 h were harvested using TrypLE™ Express solution (Thermo Fisher Scientific) and resuspended in a serum-free medium at 2 ×10⁵ cells/mL. Cells (2 × 10⁴ cells/well) were added to the wells of collagen I, collagen IV, or poly-d-lysine/laminin-coated 96-well culture plates (Corning Life Sciences, Kennebunk, ME, United States). After incubation for 30 min, the cells were washed three times with phosphate-buffered saline to remove unbound floating cells, and the attached cells were stained with a crystal violet solution (0.2% crystal violet in 20% methanol) for 30 min at room temperature. After washing with distilled water, stained cells were dissolved in 200 μl of 1% sodium dodecyl sulfate solution, and the absorbance at 590 nm was measured using a SpectraMax3 microplate reader (Molecular Devices, LLC).

iPSCs and iPSCs-Diff were seeded on 12-well culture plates, incubated overnight, and then treated with increasing concentrations of BV (0–5 μg/mL), MLT (0–5 μg/mL), apamin (0–100 μg/mL), and PLA2 (0–100 μg/mL). After 24 h, culture supernatants were collected and measured for lactate dehydrogenase (LDH) activity using Cytotoxicity Detection Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. To examine cell viability, treated cells were washed with PBS two times, and then stained with crystal violet solution (0.2% crystal violet in 20% methanol) for 30 min. After washing with distilled water thoroughly, cells were photographed and solubilized with 1% sodium dodecyl sulfate (SDS) solution. The absorbance was measured at 590 nm using a SpectraMax3 microplate reader (Molecular Devices, Sunnyvale, CA, USA). To examine selective elimination ability of BV, iPSCs cultured in mTeSR1 on Matrigel-coated 12-well culture plates were labeled with 20 μM CellTracker Green CMFDA dye (Thermo Scientific, Waltham, MA, USA) at 37 °C for 30 min. The residual fluorescence was washed out with PBS, and then un-labeled iPSCs-Diff were added and co-cultured with iPSCs in mTeSR1. After incubating in the presence or absence of BV, cells were observed under an inverted microscope and a fluorescence microscope.