Bend 3 cell line

BEND.3 cell line was purchased from American Type Culture Collection. Primary astrocytes were prepared from newborn ICR mice (within 24 hours after birth). BEND.3 cells and astrocytes were cultured in Dulbecco's modified Eagle medium (DMEM, containing 4 mmol/L L‐glutamine) supplemented with 10% fetal bovine serum (FBS; GIBCO). The culture medium was renewed every three days. For the following experiments, BEND.3 cell and the pure secondary astrocytes were then cultured in L‐glutamine‐free medium or 2 mmol/L L‐glutamine or 2 mmol/L L‐glutamine + 15 μmol/L Apoptozole for 24 hours after 5 hours of OGD and blank group was cultured in L‐glutamine‐free medium without OGD.
Primary neurons were obtained from embryos of pregnant ICR mice (16 days). Cells were seeded on Poly‐D‐lysine (Sigma‐Aldrich) coated culture flasks and grown for 4 hours in DMEM before changing into Neurobasal (Gibco) containing 2% B27 (Gibco) and 0.5 mmol/L L‐glutamine. The culture medium was half‐renewed every three days, and mature neurons were obtained from 6th day to 10th day. The mature neurons were subjected to OGD for 1 hour to induce cell injury and consequently treated the injured neurons with 1:1 mixture of fresh complete Neurobasal without L‐glutamine and CM from 0 or 2 mmol/L L‐glutamine or 2 mmol/L L‐glutamine + 15 μmol/L Apoptozole‐treated OGD astrocytes or OGD‐BEND.3 for 24 hours.

The bEnd.3 cell line, immortalized mouse brain endothelial cells, was obtained from American Type Culture Collection (Manassas, VA, USA). bEnd.3 cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium with 4500 mg/L d-glucose, 110 mg/L sodium pyruvate, 1.5 g/L sodium bicarbonate, and l-glutamine; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Mediatech Inc., Manassas, VA, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Welgene, Korea). bEnd.3 cells were maintained in a humidified incubator at 37 °C with 5% CO₂ and 95% air. All experiments were carried out when the density of cells was 90–100%.

The PC12, BV2, SH-SY5Y, and Neuro-2a cell lines were bought from Procell Life Science & Technology Co., Ltd. The bEnd.3 cell line was provided by the American Type Culture Collection (ATCC). The HT-22 cell line was obtained from Sunncell Biotech Inc. The PC12 cell line and Neuro-2a cell line were respectively cultured in the RPMI 1640 medium and MEM medium. The BV2, SH-SY5Y, HT-22, and bEnd.3 cell lines were grown in the DMEM medium.

The bEnd.3 cell line was purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C under 5% CO₂ [38 (link)]. PP2A/A, B, C subunit and endoglin expression plasmids were purchased from Shanghai Genechem Co. For transient transfection, the cells were transfected with the corresponding plasmid using the Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer's instructions.

The bEnd.3 cell line, immortalized mouse brain endothelial cells, was obtained from American Type Culture Collection (Manassas, VA, USA). bEnd.3 cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium with 4500 mg/L d-glucose, 110 mg/L sodium pyruvate, 1.5 g/L sodium bicarbonate, and l-glutamine; Welgene, Daegu, Korea), supplemented with 10% fetal bovine serum (FBS; Mediatech Inc., Manassas, VA, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Welgene, Gyeongsan, Korea). bEnd.3 cells were maintained in the incubator at 37 °C, with 5% CO₂ and 95% air. All experiments were carried out when the density was 90–100%.