BV-2 cells (a microglial cell line) were purchased from the ATCC. BV-2 cells were cultured in DMEM and supplemented with 10% FBS and 1% penicillin/streptomycin. BV-2 cells were treated with different concentrations of PDGF-BB (20 and 50 ng/mL) for 3 days. The culture medium was collected and centrifuged at 6000 rpm for 5 min. Then, the supernatant was collected and filtered by Millipak® 0.22 μm filter. Next, the CM was stored at -80℃ for further study.
The human umbilical vein endothelial cell lines (HUVECs) was purchased from ATCC and cultured in 6 wells plate with DMEM, 2% FBS, and 1% penicillin/streptomycin. HUVECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or with 1 mL of CM + 1 mL of growth medium for 1 d and harvested for further analysis.
Human brain microvascular endothelial cells (HBMECs) were obtained from Dr. Tianshi Que and purchased from Cell Systems (Seattle, WA, USA). They were cultured in 6-well plates in complete classical medium (Cell Systems) supplemented with 10% serum and culture boost (Cell Systems). HBMECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or combined with ALPL inhibitor (SBI-425, 50 µM, HY-124,756, MCE) for 1 d and harvested for further analysis.
The human umbilical vein endothelial cell lines (HUVECs) was purchased from ATCC and cultured in 6 wells plate with DMEM, 2% FBS, and 1% penicillin/streptomycin. HUVECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or with 1 mL of CM + 1 mL of growth medium for 1 d and harvested for further analysis.
Human brain microvascular endothelial cells (HBMECs) were obtained from Dr. Tianshi Que and purchased from Cell Systems (Seattle, WA, USA). They were cultured in 6-well plates in complete classical medium (Cell Systems) supplemented with 10% serum and culture boost (Cell Systems). HBMECs were treated with IL-1β (10 ng/mL, 10,139-HNAE, Sino Biological) or combined with ALPL inhibitor (SBI-425, 50 µM, HY-124,756, MCE) for 1 d and harvested for further analysis.