Glutamate dehydrogenase activity assay kit

According to the manufacturer instructions (Glutamate Dehydrogenase Activity Assay kit, Abcam, ab102527), cells (1.10⁶ cells) were homogenized in 200μL of assay buffer on ice and centrifuged at 13 000g 4°C for 10 min. Protein conc entration was determined by Bradford assay. Samples, normalized to total protein (100μg), were incubated with kit reagents for 1 hr at 37°C, and absorbances were measured at 450nm.

One control and one cKO mouse, aged P50–55 and sex matched, were sacrificed, and brain tissues processed, in parallel on each day of an experiment. Animals were deeply anesthetized with an intraperitoneal injection of Euthasol. Animals irresponsive to toe pinches were perfused with ice-cold PBS. Brains were rapidly extracted and midbrains were dissected out on ice, then flash frozen in pre-chilled isopentane. Tissue samples were then prepared according to kit manufacturers’ instructions. The following kits were used for detection of metabolites: Biovision Glutamate Colorimetric Assay Kit (cat. K629), Biovision Glutamine Colorimetric Assay Kit (cat. K556), Biovision Ammonia Colorimetric Assay Kit (cat. K370), Abcam Glutamate Dehydrogenase Activity Assay Kit (for NADH, cat. Ab102527), Bioassays QuantiChrom Glutathione (GSH) Assay Kit (cat. DIGT-250). Kit reactions produced stable colorimetric signals that were measured as absorbance in a specific wavelength by a plate reader (BioTek Snyergy H1 Hybrid Reader). Plates included vehicle wells with no sample for background subtraction. For each tissue sample, two technical replicates were included per well and averaged. No sex differences were observed (e.g., Glutamine t = 1.106, p = 0.3839).