Ldh activity assay kit

The cells were evaluated for viability using Live/Dead staining kit (Molecular Probes). After 72 h, the cells were incubated in DMEM-F12 containing 1 μM calcein-AM (green) and 2 μM ethidium homodimer I (red) for 30 min. The samples were imaged under a fluorescent microscope (Olympus IX70). With ImageJ software, the viability was analyzed and calculated as the percentage of green intensity over total intensity (including both green cells and red cells). For MTT assay, the replated neural cells were incubated with 5 mg/mL MTT (Sigma) solution. The absorbance was measured at 500 nm using a microplate reader (Biorad). Image-iT Live Green Poly Caspase Detection kit (Molecular Probes) was used to detect the expression of caspases. The replated cells were incubated for 1 h with the fluorescent inhibitor of caspases reagent and analyzed by a fluorescence microscope.^{40 (link),41} The cytotoxicity of cells was assessed using LDH activity assay kit (Sigma, MAK066). Briefly, a total volume of 100 μL of spent medium and LDH reaction mixture was mixed well, and the initial absorbance at 450 nm was measured using a microplate reader (Bio-Rad iMark). The mixture was incubated at 37 °C, and a measurement was taken every 5 min. The LDH activity was calculated through the subtraction of final and initial measurements in comparison to the standard curve.

Serum LDH activity was measured by the LDH Activity Assay Kit (Sigma-Aldrich, St. Louis, MO; Catalog Number: MAK066) according to the manufacturer’s instructions.