Anti cd146 antibody coated microbeads

EMSCs were isolated by sequential beading with magnetic beads coated with anti-CD140b and anti-CD146 antibodies as described [5 (link)]. The stromal cells were first successively incubated with PE-conjugated anti-CD140b antibody (10 μl/10⁶ cells, R&D Systems, Minneapolis, MN, USA) for 45 min at 4 °C and then with anti-mouse IgG1-coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4 °C before they were loaded onto Miltenyi MS columns with a magnetic field to collect the CD140b⁺ cells. The stromal CD140b⁺ population was cultured in fibronectin-coated dishes containing growth medium at 37 °C in 5% CO₂ for 7–10 days to allow detachment of the microbeads during cell expansion. They were then trypsinized and incubated with anti-CD146 antibody-coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4 °C. The CD140b⁺CD146⁺ cells (eMSCs) were obtained after magnetic column separation.

Two successive magnetic bead selections were carried out to isolate the CD140b⁺ and CD146⁺ cells (eMSC) [28 (link)]. Firstly, stromal cells were incubated with phycoerythrin (PE)-conjugated anti-CD140b antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at 4 °C followed by another 15 min incubation with anti-mouse IgG1 magnetic microbeads (Miltenyi Biotech, San Diego, CA, USA). The cell suspensions were then loaded onto MS columns (Miltenyi Biotech, San Diego, CA, USA) with a magnetic field to separate the CD140b⁺ cells, which were cultured for 7–10 days to allow degradation of the microbeads during cell expansion. The cells were then trypsinized and incubated with anti-CD146 antibody-coated microbeads (Miltenyi Biotech, San Diego, CA, USA) for 15 min at 4 °C to obtain the CD140b⁺CD146⁺ cells for experimentation [28 (link)]. Normal humidified cell incubator with 5% CO₂ was used for normoxic cultures (21% O2). For hypoxic cultures (2% O₂), the cells were placed into a sealed chamber (Thermo Fisher Scientific, Waltham, MA, USA), which was flushed with a gas mixture of 2% O₂, 5% CO₂, and 93% N₂ (v/v) at 37 °C.

Two positive selections using magnetic microbeads were used for the acquisition of CD140b⁺CD146⁺ eMSC (19 (link)). Firstly, in vitro-expanded stromal cells were incubated with PE-conjugated anti-CD140b antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at 4° and then with antimouse IgG1-coated magnetic microbeads (Miltenyi Biotec Inc.) for another 15 min. After applying the cell suspension to MS columns (Miltenyi Biotec Inc.) in the magnetic field, CD140b⁺ cells were obtained and further cultured for 7 to 10 days to allow microbead degradation during cell expansion. For the second round of selection, cells were incubated with anti-CD146 antibody-coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4°. CD140b⁺CD146⁺ eMSC were then obtained from column separation. Stromal cells at passages 1–3 were employed in this study.