Dose-Dependent Screening of RSL3 in Motor Neurons

Dissociated motor neuron cells were plated onto two PLO‐coated 96‐well plates (cat. 3904; Corning, Teterboro, NJ, USA) at a density of 150 000 cells per well in a volume of 100 μL. These are the assay plates. Two days later, two empty 96‐well polypropylene plates (cat. 3365; Corning) were labeled as ‘high’ and ‘low’ and filled with 100 μL of motor neuron media except column 4 of ‘high’ plate where 200 μL of 4 μm RSL3 was transferred. Then, twofold serial dilution of the RSL3 across columns 5–11 in ‘high’ and columns 2–9 in ‘low’ was carried out by transferring 100 μL of the compound solution to the next columns successively with mixing. iNIL‐MN cells in the assay plates were treated with RSL3 in a twofold dilution series by transferring 100 μL solution from the compound plates. Finally, the assay plates were returned to the culture incubator and maintained for 24 h before starting resazurin viability assay. The final concentration of RSL3 in the assay plate starts from 2 μm, and then, twofold diluted in the next wells accordingly.