Ldh assay kit

Culture supernatants were collected 2 to 3 times a week and kept at − 80 °C until the assays were performed. All experiments were performed according to the manufacturers’ protocols. In brief, released IL-1β, IL-6, and TNFα were measured from undiluted supernatants using commercially available V-Plex rat ELISA kit (Meso Scale Discovery, Rockville, MD), and electroluminescent signals were detected by MESO QuickPlex (Meso Scale Discovery, Rockville, MD). Lower detection limit of the kit was 6.92 pg/ml for IL-1β, 13.8 pg/ml for IL-6, and 0.72 pg/ml for TNFα. Lactate dehydrogenase (LDH) was measured using LDH assay kit (BioVision, Milpitas, CA). The culture supernatants were diluted two- to threefolds with the assay buffer (BioVision), and colorimetric signals were measured at 450 nm by a microplate reader FlexStation 3 (Molecular Devices, San Jose, CA). LDH concentrations were calculated from the level of LDH activity that converts NAD to NADH. The concentration was expressed as milliunits per milliliter, in which 1 U of LDH generates 1 μM NADH per minute. Goat immunoglobulin G concentration in culture supernatants was measured using the competitive inhibition IgG ELISA kit (Cusabio, College Park, MD). The detection range of the kit was 0.146 to 37.5 μg/ml.

Supernatants of cultured RGCs were collected and measured by the LDH Assay Kit (BioVision, Mountain View, CA, USA) according to the manufacturer’s protocol. The absorbance plate reader (BioTek, Winooski, VT, USA) was used to read the amount of LDH release from the RGCs.