Anti human cd140b pe

Human brain pericytes (passage 2, three independent experiments in triplicates) were harvested (20,000 cells) and washed three times with PBS. Cells were incubated with anti-human CD140b-PE (#558821), anti-human CD31-FITC (#560984), (BD Biosciences, San Jose, CA), and anti-human CD146-FITC (#MCA2141F) (AbD Serotec) fluorochrome-conjugated antibodies, in separated tubes for 45 min at 37 °C in the dark. IgG1-PE (#555574) and FITC (#555748) mouse antibodies (BD Biosciences) were used as isotype control to set the threshold and gates in the cytometer. Finally, samples were analysed by flow cytometry with an Accuri C6 (BD Biosciences). Briefly, the region corresponding to pericytes was defined by a combination of forward (FSC) and side scatter (SSC) properties. Isotype control and unstained pericytes were used to set the background and the gate settings. Pericytes were identified according to the expression of membrane-specific antigens (CD140b, CD146, CD31) in the PE and FITC fluorescence channels. A total of 500,000–1,000,000 events were recorded for each sample and analysed with the FACS Diva software (Tree Star, Inc.).

VSMC differentiation was carried out as previously described (Patsch et al., 2015 (link)). Briefly, iPSCs were unicellularized using TrypLE Express (Gibco) and seeded onto matrigel-coated 6-well plates at a density of 3 × 10⁵ per well. After incubated in mTeSR supplemented with 10 µmol/L Y-27632 (Selleck) for one day, cells were then cultured in the N2B27 medium supplemented with 25 ng/mL BMP4 (R&D) and 8 μmol/L CHIR99021 (Selleck) for 3 days. Finally, cells were cultured in N2B27 medium supplemented with 10 ng/mL PDGF-BB (Peprotech) and 2 ng/mL Activin A (HumanZyme) for another 2 days and sorted after stained with anti-human CD140b-PE (BD biosciences, 558821, 1:200) by a flow cytometry (BD FACSAria IIIu). IgG-PE (BD biosciences, 555749) was used as an isotype control.