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Glutamate production was measured using the Glutamate Assay Kit (Sigma-Aldrich, MAK004). In brief, the supernatants of cultured neurons were deproteinized with a 10 kDa MWCO spin filter before addition to the reaction. Glutamate standard was prepared by diluting 0.1 M glutamate (provided in the kit) to a different concentration gradient. The glutamate standard and each sample were added into a 96-well plate (50 μl per well), and 100 μl reaction solution was added to each well and mixed thoroughly. The mixture was incubated at 37 °C for 60 min. The absorbance of each well was measured on a microplate reader using 450 nm as the primary wavelength. The glutamate concentration of each sample was calculated by glutamate standard curve.
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