Cd146 vioblue

Cells from the sorting procedure were centrifuged at 300g for 10 min and pellets resuspended in the appropriate fluorescent antibodies or isotype control antibodies at a concentration of 1:11 in MACS buffer. Cells were incubated in up to three antibodies for 10 min at 4 °C. After a final wash step, cells were resuspended in MACS buffer and transported directly for flow cytometry and analysed for surface marker expression using a Cyan ADP flow cytometer (Beckman Coulter, High Wycombe, UK) at the Faculty of Biology, Medicine and Health Core Facility, University of Manchester. Compensation was carried out using a bead kit (Miltenyi Biotec, Surrey, UK, 130-097-900). All antibodies and isotype controls used were obtained from Miltenyi Biotech (Surrey, UK) and are as follows with product codes: CD29-PE (130-101-275), CD34-PE-Vio770 (130-100-844), CD45-PerCP (130-098-145), CD90-FITC (130-097-930), CD146-VioBlue (130-099-678), CD271-APC (130-091-884), Mouse IgG1-PE (130-098-845), Mouse IgG2a-PE-Vio770 (130-098-564), Mouse IgG2a-PerCP (130-099-190), Mouse IgG1-FITC (130-098-847), Mouse IgG1-VioBlue (130-099-756), and Mouse IgG1-APC (130-098-846). Data were analysed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).

Flow cytometry was performed on MSCs (s.c.ASC, o.ASC, and BMSC) at passage 3. The cells were trypsinized, subsequently centrifuged at 1,400 rpm for 5 min, and washed with PBS containing 0.5% bovine serum album (Sigma-Aldrich) and 0.5 M EDTA (Lonza). The number of cells was determined by hemocytometer. A total of 2 × 10⁶ cells were incubated with different fluorochrome-conjugated anti human monoclonal antibodies for 30 min. The following CD surface markers were tested: CD45/APC-Cy7, CD90/APC, CD105/FITC, CD73/PE, CD235a/PE-Cy5, CD34/AF700 (BD Biosciences, San Jose, CA, USA), CD31/PE-Cy7 (BioLegend), CD33/PC5, CD14/PC5 (Beckman Coulter, Brea, CA, USA), and CD146/VioBlue (Miltenyi Biotec). For each antigen 10,000 events were collected on an LSRII flow cytometer (Becton Dickinson). Analysis was conducted using Flow Jo software (Tree Star).