Type 1 or 4 collagen

Immortalized mouse BECs of the bEnd3 cell line were obtained from the Shanghai Bioleaf Biotech Co., Ltd. Primary human brain microvascular endothelial cells (HBMECs) were purchased from Cell Systems (Kirkland, WA, USA). Cells (bEnd3 and HBMECs) were grown in six-well plates pre-coated with type I or IV collagen (10 μg/mL, Sigma, for 2 h at 37 °C). The culture medium was endothelial basal medium (EBM-2) (Lonza, CC-3156) supplemented with 10% FBS (Gibco), ascorbic acid, L-glutamine, penicillin/streptomycin, and human basic fibroblast growth factor (bFGF) (all from Sigma). Cells were maintained in a humidified incubator at 37 °C and 5% CO2, and the medium was changed every 48 h.

Immortalized mouse brain endothelial cells (BECs) of the bEnd3 cell line were obtained from Shanghai Bioleaf Biotech Co., Ltd. Cells were grown on six-well plates pre-coated with type I or IV collagen (10 μg/ml, Sigma, for 2 h at 37 °C) cultured in endothelial basal medium (EBM-2) (Lonza, CC-3156) supplemented with 10% FBS (Gibco), ascorbic acid, L-glutamine, penicillin/streptomycin, and human basic fibroblast growth factor (bFGF) (all from Sigma). Cells were maintained in a humidified incubator at 37 °C and 5% CO₂, and the medium was changed every 48 h.