Glutamine assay kit

The glutamine concentration in hearts was analyzed using a Glutamine Assay Kit (Abcam, ab197011) according to the manufacturer’s protocols. Briefly, 10–20 mg of fresh left ventricle tissue was homogenized in ice-cold Hydrolysis Buffer, and the insoluble fraction was removed by centrifugation. The supernatant was added to perchloric acid (PCA). After 5 minutes incubation on ice, the samples were centrifuged and the supernatants were transferred into new tubes. To remove excess PCA, potassium hydroxide was added to the supernatant and the precipitated PCA was removed by centrifugation. A microplate reader was used to measure the absorbance at OD 450 nm. The glutamate concentration in hearts was analyzed using a Glutamate Assay Kit (Abcam, ab83389) according to the manufacturer’s protocols. Briefly, 10–20 mg of fresh left ventricle tissue was homogenized in ice-cold Assay Buffer, and the insoluble fraction was removed by centrifugation. The supernatants were added into a 96-well microplate for analysis. A microplate reader was used to measure the absorbance at OD 450 nm.

Glutamate and glutamine level in mice liver tissue was assessed measured using commercial Glutamate Assay kit (Abcam, ab138883) and Glutamine Assay kit (Abcam, ab197011), respectively. All procedures were according to the manufacturer’s protocols.

The glutamine concentrations were measured using a glutamine assay kit (Abcam). Briefly, cells (2 × 10⁶) were washed twice with cold phosphate-buffered saline, resuspended in ice-cold hydrolysis buffer, homogenized, and centrifuged at 4 °C at 10,000 × g for 10 min to collect the supernatant. Ice-cold perchloric acid (PCA) was used for deproteinization and KOH (2 M) was used to precipitate excess PCA. Then, samples were incubated with glutamine-reaction mix at 37 °C for 60 min, and absorbance at 450 nm was recorded on a microplate reader.

Glutamine uptake, glutamate production, and α-ketoglutarate production were analyzed to evaluate cell glutamine metabolism according to the protocols of Glutamine Assay Kit (Abcam), Glutamate Assay Kit (Abcam), and α-ketoglutarate Assay Kit (Abcam), respectively.