Humedia eg

To prepare CD31⁺F4/80⁺ cells from E10.5 mouse brains, WT or Egfp Tg brains were mechanically triturated with a pipette in PBS on ice. To fractionate CD31⁺F4/80⁺NG2⁻ cells from yolk sacs, E10.5 R26-mCherry mouse yolk sacs were digested using Accutase (Nacalai Tesque) with 0.2% collagenase (Nacalai Tesque) and 0.005% trypsin inhibitor (Nacalai Tesque) for 1 hour at 37 °C then gently triturated with a pipette on ice. These cells were rinsed in PBS and then blocked with 3% normal mouse serum (Dako, Glostrup, Denmark) on ice for 10 min. Blocked cells were simultaneously stained with a PE-conjugated anti-F4/80 antibody (Bio-Rad Laboratories), an APC-conjugated anti-CD31 antibody (BD Biosciences) and, if needed, an Alexa488-conjugated anti-NG2 antibody (Merck Millipore) on ice for 15 min. Labeled cells were sorted using a FACSAria cell sorter (BD Biosciences). CD31⁺F4/80⁺ cells sorted from WT embryonic brains were seeded on fibronectin- or collagen type I-coated 8-well slides (BD Biosciences) and cultured in HuMedia-EG (KURABO, Osaka, Japan) for the durations indicated. CD31⁺F4/80⁺NG2⁻ cells sorted from R26-mCherry mouse yolk sacs were co-cultured with b.End5 cells and mouse neural stem/progenitor cells for the durations indicated.

Bovine brain microvasculature endothelial cells (BBMCs) and human umbilical vein endothelial cells (HUVECs) were used. Cultures were grown for 24 h at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ before use in experiments. BBMCs were plated in a 10-cm dish coated with collagen in containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic-antimycotic (Invitrogen), and 10 ng/mL basic FGF (Roche Applied Science, Penzburg, Germany). HUVECs were also plated in a 10-cm dish coated with collagen in medium (Humedia-EG; Kurabo, Osaka, Japan).
An adenovirus-mediated reporter system was prepared. HUVECs were transfected with purified adenovirus, including miRNAs targeting β-arrestin-2 and control miRNA, and evaluated after 2 days. The miRNA sequence for the knockdown of β-arrestin-2 was 5′-TGCTGATACCTGGTCATCTTGTTCGAGTTTTGGCCACTGACTGACTCGAACAATGACCAGGTAT-3′. The sequence of control miRNA was LacZ-specific miRNAs. HUVECs samples were collected after 24 h in palmitic acid (WAKO) in 100% Ethanol conjugated to 10% bovine serum albumin.

MS-1 (mouse pancreatic islet endothelial cell line) was purchased from the ATCC (Manassas, VA) and maintained in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 5% heat-inactivated-FBS and p/s. HUVECs (human umbilical vein endothelial cells) were purchased from Kurabo (Osaka, Japan) and maintained in HuMedia-EB2 supplemented with HuMedia-EG (Kurabo).