Measuring Changes in Mitochondrial Metabolism in Hypoxic RPE Using Resipher System

To begin, assemble the sensing lid with the cell culture plate in the hood and transfer them into the hypoxia chamber. Set up the sandwich in the hypoxia chamber. After that, place a Petri dish with sterile water in the hypoxia chamber to help maintain humidity.

Finally, place a portable oxygen sensor in the hypoxia chamber. Then, seal the hypoxia chamber. Ensure the USB cable is coming out of the hypoxia chamber, and the hole where the cable exits is sealed with putty or silicone grease.

Connect the hypoxia chamber to a gas cylinder with a hypoxic concentration of oxygen and a standard cell culture concentration of carbon dioxide. Slowly exchange the air in the hypoxia chamber with the 1%oxygen and 5%carbon dioxide air from the cylinder until the oxygen sensor shows the desired oxygen concentration, usually between 1%and 10%Now, close the inlet and outlet valves of the hypoxia chamber and transfer the entire chamber into the incubator. Then set up and start the experiment.

Media equilibrated with atmospheric oxygen took longer to reach equilibrium at higher volumes. The hypoxia chamber showed a slow leak, causing oxygen levels to approach atmospheric concentration by 30 hours. Oxygen solubility was identical between fresh and conditioned media, indicating that media composition changes over time did not affect oxygen solubility.