Nadph quantitation fluorometric assay kit

NAD⁺ levels were assessed using NAD⁺/NADH Quantification Colorimetric Kit (#K337-100, Biovision), NADPH levels were assessed using NADPH Quantitation Fluorometric Assay Kit (#K349-100, Biovision), and ATP levels were assessed using ATP Colorimetric/Fluorometric Assay Kit (#K354-100, Biovision) following the protocols provided by the supplier. Results were normalized with protein concentration and measured using SpectraMax i3.

The presence of various enzymes and proteins could interfere with the detection of metabolites in cultured astrocytes and consequently be removed using the Deproteinizing Sample Preparation Kit (Biovision, USA). A glycogen assay kit (Biovision) was used to detect glycogen levels in cultured astrocytes. ROS levels were determined using the ROS Detection Assay Kit (Biovision). A glucose-6-phosphate colorimetric assay kit (Biovision) was used to detect the glucose-6-phosphate levels. NADPH levels were determined with the NADPH Quantitation Fluorometric Assay Kit (Biovision). Glutathione levels were analyzed with the Glutathione Fluorometric Assay Kit (Biovision).
To determine the glycogen levels in the brain, the mice were decapitated, and brain tissues in the penumbra were immersed in liquid nitrogen. Then, the brain tissues (10 mg) were homogenized with 200 μL 30% KOH on ice, and the homogenates were boiled for 10 min to inactivate enzymes. The boiled samples were centrifuged at 12,000 rpm at 4 °C for 10 min to remove insoluble sediments. Then, the supernatant was ready for the assay using a glycogen assay kit (Biovision), and the results were normalized to the protein levels in homologous samples.