Seahorse XF real-time ATP rate assay

Agilent Seahorse XFe96 analyzer was used to measure multiple parameters including oxygen consumption rate (OCR), total ATP production by quantifying ATP production rate from both glycolytic and mitochondrial pathways and ATP rate index. Briefly, primary RGCs were isolated from post-natal day 4–6 rat pups. The RGCs were seeded RGC medium in a poly-D-lysine coated seahorse plate and maintained in culture for one week to allow neurite outgrowth. The RGCs were either untreated or treated with ET-1 for 4 h or 24 h in trophic factor-free medium. A day prior to the experiment seahorse sensor cartridge was hydrated with water and incubated in a non CO₂ incubator at 37 °C. Two hours before the experiment, the water was replaced with a seahorse calibrant. On the day of the experiment, seahorse XF DMEM (pH=7.4) was supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose (Agilent, USA). The RGC medium was then replaced with 180 µl of Seahorse XF base medium and the cells incubated in a non CO₂ incubator at 37 °C for 1 hour. During this incubation period, oligomycin and Rotenone/antimycin (Agilent, USA) were prepared in the seahorse medium to achieve final concentrations of 1.5 μM and 0.5 μM respectively when injected. From these stock solutions, 20 μl and 22 μl of Oligomycin and rotenone/antimycin respectively were then loaded into the drug delivery ports A and B of the hydrated sensor cartridge and loaded into the seahorse XF analyzer to calibrate for 30 minutes. The calibration plate was replaced with the cell culture plate and oxygen consumption (OCR) and extracellular acidification rate (ECAR) was monitored following the sequential injection of Oligomycin and rotenone/antimycin with each cycle set as 3 min mix, 2 min delay and measure for 3 minutes. According to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA), the following equation was used to calculated mitochondrial ATP production:

The ATP production rate in ET-1 treated RGCs was compared to that of untreated RGCs for two time points of ET-1 treatment (4 h and 24 h). Data was normalized to the cell number of each well by Calcien AM assay.