C7026

Primary astrocytes were plated at a density of 2,000 cells/well in XF96 plate. The procedures of the glycolytic rate assay were conducted according to the user guide of Agilent Seahorse XF Glycolytic Rate Assay Kit (103344-100, Agilent Technologies). The day before assay, assay medium was prepared by supplementing base medium with 10 mM glucose, 1 mM pyruvate, 2 mM glutamine and 5.0 mM HEPES (pH 7.4). After 72 h stimulation with corticosterone and fluoxetine, cells were washed with warm assay medium for two times and the XF96 cell culture plate was calibrated in a non-CO₂ incubator at 37°C for 1 h. After calibration, OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were measured by seahorse analyzer. Rotenone/antimycin A (0.5 μM) and 2-DG (5 mM) were added to detect the compensatory glycolysis and post 2-DG acidification. GlycoPER (glycolytic proton efflux rate) was calculated according to the values of OCR and ECAR. After seahorse glycolytic rate assay, the DNA level per cell was detected by commercial kit (C7026, Invitrogen). The results of glycoPER were normalized by cell content (the ratio of DNA level).

DAPI staining and residual DNA quantification were used to evaluate the residual DNA which was an important index for dECM. For DAPI staining, the samples were fixed with 4% formaldehyde for 15 min and rinsed with PBS. DAPI solution (#4083, Cell Signaling Technology) at the concentration of 1 μg/mL was added, and the samples were incubated for 5 min at room temperature avoiding light. After PBS washing, the samples were coated with anti‐fade mounting solution and imaged under a fluorescence microscope (Eclipse Ti2, Nikon).
The content of residual DNA was quantified by a cell proliferation assay kit (C7026, Invitrogen) according to the manual. Native cells were washed with PBS, frozen, and stored at −80°C. The frozen cells and dECMs were thawed at room temperature and incubated in CyQUANT® GR dye/cell‐lysis buffer for 2–5 min, protected from light. The samples were measured using a fluorescence microplate reader with filters for 480 nm excitation and 520 nm emission maxima. The standard curve was prepared with λDNA (provided by the kit). The DNA content of dECM divided by the DNA content of native cells was defined as relative DNA percentage.