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

## TL;DR

This paper provides a detailed protocol for measuring glucose metabolism in mouse retinas and RPE-choroid tissue using stable isotope labeling and GC-MS.

## Contribution

The protocol introduces a method to assess retinal metabolic flux using 13C6-glucose and GC-MS for isotopic labeling analysis.

## Key findings

- The protocol enables tracking of glucose metabolism in retinal and RPE-choroid tissues.
- It uses GC-MS to determine isotopic labeling in glycolysis and the TCA cycle.
- The method is adaptable for various tissues, animal models, and genetic conditions.

## Abstract

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

•Guidelines for dissecting mouse retina and RPE-choroid•Steps for incubating tissues with 13C-glucose•Instructions for extracting metabolites after incubation•Procedure for analyzing isotopic enrichment by GC-MS

Guidelines for dissecting mouse retina and RPE-choroid

Steps for incubating tissues with 13C-glucose

Instructions for extracting metabolites after incubation

Procedure for analyzing isotopic enrichment by GC-MS

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for evaluating glucose metabolism in mouse retinas and 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.