Measurement of ATP Levels in Brain Tissue

After ¹H-MRS, the mice were euthanized with sodium pentobarbital (80 mg/kg, i.p.) and decapitated. The brain was removed from the skull and the brain regions of PFC and CPU were dissected out quickly and frozen in liquid nitrogen until use for ATP measurement which was performed with an ATP Assay Kit (S0026; Beyotime Biotechnology, Shanghai, China) following the operation steps detailed by the manufacturer as follows: (1) sample preparation: the tissue sample was homogenized in the 1 × ATP detection sample buffer provided in the assay kit. After centrifugation (12,000 g, 4°C) for 5 minutes, the supernatant was collected and diluted with the buffer in a pre-chilled polypropylene tube on ice. (2) prepared ATP detection standard solutions by diluting the 5 mM ATP solution in 1 × ATP detection sample buffer in wells A-G. The final ATP concentrations in the wells were in the range of 5 nM –5 mM at the same volume of 200 μL. (3) performing assay: added 100 μL freshly prepared reaction mixture, which was consisted of 1 × ATP detection assay buffer, Dd-Luciferin and Luciferase, to wells on a 96 -wells plate, and kept the plate at room temperature (24°C) for 3 min. Added 20 μL of 1 × ATP detection sample buffer to two blank wells, ATP detection standard to wells A-G (standard wells), and sample solution to designated wells on the plate. Cover the plate with the plate cover and incubated at room temperature for 5–10 min. Removed the plate cover and read the luminescence in a Tecan microplate reader (M20Pro, Tecan Trading AG, Switzerland). Calculated the ATP concentration in each sample after obtaining the average luminescence of blank, standard, and sample wells and subtracting the average luminescence of blank wells from the average luminescence of standard and sample wells. The ATP concentrations of all samples were within the standard curve of the assay.