Protocol to assess retinal metabolic flux of mice via stable isotope-resolved metabolomics

Here, we present a protocol for evaluating glucose metabolism in mouse retinas and retinal pigment epithelium (RPE)-choroid tissue by tracking the incorporation of ¹³C₆ from U-¹³C₆-glucose with gas chromatography-mass spectrometry (GC-MS). We describe steps for incubating tissues in Krebs-Ringer bicarbonate solution and homogenizing tissues. We then detail procedures for extracting metabolites and determining isotopic labeling of intermediates in glycolysis and the tricarboxylic acid (TCA) cycle using GC-MS. The approach has been adapted to study glucose metabolism in various tissues, animal models, and genetic conditions. For complete details on the use and execution of this protocol, please refer to Nolan et al.¹.