Protocol for real-time measurement of mitochondrial respiration in the mouse ocular posterior pole using a Seahorse XFe24 analyzer.

During aging and in retinal degenerative diseases, vulnerable retinal pigment epithelial (RPE) cells are subject to mitochondrial dysfunction, creating a need for accessibility to tools which can facilitate assessment of the ocular posterior pole bioenergetics. Here, we present a protocol for quantifying mitochondrial respiration in the posterior eye cup (RPE-choroid-sclera) of young and old mice. We describe steps for eye cup dissection, optimization of tissue size, drug concentrations, and cycle conditions using the XF Cell Mito Stress Test.