2.10. Cell Isolation and Treatments

Primary cortical neurons were isolated from the cortices of E16-E18 mice according to previous studies [29]. Briefly, the cortex was dissected and digested in 0.125% trypsin at 37°C for 10 min, followed by the neutralization in neurobasal medium (GIBCO). Next, the collected suspensions were centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in fresh neurobasal medium containing 10% B27, 0.5 μmol/L glutamine, and 25 μmol/L glutamate, followed by the filtration using a 40 μm strainer. For oxygen glucose deprivation/reperfusion (OGD/R) stimulation, the cells were rinsed with phosphate-buffered saline for 3 times and incubated with glucose-free HBSS buffer at 95% N₂/5% CO₂ at 37°C for 4 h and then placed in the fresh neurobasal medium under normal condition for additional 24 h. Neurons cultured in normal oxygen- (95% air, 5% CO₂) conditioned fresh neurobasal medium for the same periods were used as controls (Ctrl) [40]. For USP29 silence, the primary cortical neurons were isolated from USP29^flox/flox mice and then infected with AdCre for 4 h at the multiplicity of infection (MOI) of 20 to knock down the endogenous USP29, followed by the incubation in fresh neurobasal medium for additional 48 h before OGD/R stimulation. For USP29 overexpression, the wild type primary cortical neurons were infected with AdUSP29 at the MOI of 10 for 4 h. To knock down the endogenous SIRT1 or BMAL1, the primary cortical neurons from USP29^flox/flox mice were incubated with siSIRT1 (50 nmol/L) or siBMAL1 (50 nmol/L) using Lipo6000™ Transfection Reagent for 4 h and placed in fresh neurobasal medium for additional 24 h before AdCre infection [41, 42]. For p53 inhibition, the wild type primary cortical neurons were pretreated with PFT-α (10 μmol/L) for 12 h before OGD/R stimulation [31]. To inhibit miR-34a, the cells were treated with miR-34a antagomir (50 nmol/L) using Lipo6000™ Transfection Reagent for 4 h and kept for additional 24 h before AdUSP29 infection.