Endogro mv complete medium

The immortalized human cerebral microvascular endothelial cell line (hCMEC/D3) was obtained from Cedarlane (Tebu-Bio, France) and maintained in EndoGRO™-MV Complete Medium (cat# SCME004, EMD Millipore, USA) supplemented with 1ng/mL FGF-2 (cat# GF003, EMD Millipore) and antibiotics at 37°C in 5% CO2. Cells were regularly tested for mycoplasma contamination and phenotypic changes.

Immortalized human cerebral microvascular endothelial cell line (hCMEC/D3) was purchased from Merck-Millipore (Darmstadt, Germany). All cultureware used for hCMEC/D3 cells was coated with rat tail collagen type I (Gibco) diluted in phosphate buffered saline (PBS) 1:20 and incubated at 37 °C for at least 1 hour. Cells were maintained in EndoGRO-MV complete medium (Merck-Millipore) containing EndoGRO basal medium, 5% FBS, L-glutamine (10 mM), EndoGRO-LS supplement (0.2 %), heparin sulphate (0.75 U/ml), ascorbic acid (50 μg/ml), hydrocortisone hemisuccinate (1 μg/ml), recombinant human epidermal growth factor (5 ng/ml) and additionally supplemented with freshly added recombinant human fibroblast growth factor-basic (1 ng/ml; Gibco). hCMEC/D3 cells used for the experiments were between passages 6 and 11. All cell lines were maintained at 37 °C in a humidified atmosphere containing 5 % CO 2 . The medium was changed twice a week. For passaging, cells were washed once with PBS, harvested with 0.25 % trypsin/ 1 mM EDTA solution (Gibco), resuspended in culture medium and plated onto cell culture flask for expansion.

Immortalized human umbilical vein ECs (CI-huVECs, InSCREENeX, Braunschweig, Germany) and human cerebral microvascular ECs (hCMEC/D3; Merck Millipore, Darmstadt, Germany) were cultured at 37°C and 5% of CO 2 in EC growth medium (ECGM, PromoCell, Heidelberg, Germany) supplemented with 10% of fetal calf serum (FCS, Thermo Fisher Scientific, Waltham, MA, USA) or in EndoGRO-MV complete medium (Merck Millipore) supplemented with 1 ng/mL of FGF-2 (PromoCell) and 5% of FCS, respectively. For rescue experiments, human plasma fibronectin (F2006, Sigma-Aldrich, St. Louis, MO, USA), human cellular fibronectin (F2518, Sigma-Aldrich), a proteolytic human plasma fibronectin 70 kD fragment (F0287, Sigma-Aldrich), a human fibronectin 120 kD cell attachment fragment (Part Number 175, YO Proteins, Ronninge, Sweden), human type IV collagen (C5533, Sigma-Aldrich), recombinant periostin (rPOSTN; RPH339Hu01, Cloud-Clone Corp., Katy, TX, USA), and recombinant fibulin-5 (rFBLN5; 9006-FB-050, R&D Systems, Minneapolis, MN, USA) were either supplemented to the ECGM or used to coat cell culture plates with the indicated concentrations. If not stated otherwise, the cells were cultured for 48 hours with ECM protein supplementation. Cells cultured on uncoated, tissue culture treated plates served as controls.

Human cerebral microvascular endothelial cells (hCMEC/D3) were obtained from the Institut Cochin (INSERM, Paris, France). Cells at passages between 27 and 37 were grown on tissue culture
flasks, covered with 0.1 mg/ml rat tail collagen type 1, in EndoGRO-MV complete medium (Merck Millipore) supplemented with 1 ng/ml basic FGF and 1% Penicillin-Streptomycin (Life Technologies). Cells were seeded at a density of 24,000-33,000 cells/cm 2 and cultured at 37°C, 5% CO 2 . For flow cytometry analysis, cells were cultured on type 1 collagen-coated 12-well plates; confluent hCMEC/D3 monolayers were obtained typically by day 3.