LDH cytotoxicity assay

Lactate dehydrogenase (LDH) is known as the signal representing the integrity of cells’ plasma membranes. First, the iPSC–RGCs were maintained in 24 wells for 15 days and then added to 0/30/50/70 mM NaCl combined with or without a neurobasal medium containing 5 μM H89 for 24 h. The sample medium was collected and applied to the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) for measuring LDH activity. According to the manufacturer’s instructions, 50 μL of the sample medium and the positive control were transferred to a 96-well flat-bottom plate and gently mixed with substrate mixtures at room temperature for 30 min. Each of the reactions was terminated using 50 μL of stop solution. The LDH activity was represented by highly colored and soluble formazan with absorbance at 490 nm, which was determined by Infinite M1000 Pro plate reader (Tecan). To specifically calculate the LDH release levels in each sample, we harvested the remaining cells for protein measurement. The cells were lysed in 1× RIPA buffer and the protein concentration was measured through Braford’s method. After the protein concentration of each sample was found, the LDH release level was eventually determined as OD values/protein concentrations.