Anti mouse igg1 magnetic microbeads

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.

Isolation of eMSCs (CD140b⁺CD146⁺ cells) was conducted with two separate positive magnetic bead selections [16 (link)]. Stromal cells were incubated with phycoerythrin (PE)-conjugated anti-CD140b antibody at 4 °C for 45 min. The cells were then incubated with anti-mouse IgG1 magnetic microbeads (Miltenyi Biotech) at 4^oC for 15 min. The CD140b⁺ cells were collected using the Miltenyi columns with a magnetic field and cultured for 7 to 10 days in growth medium to allow degradation of the microbeads during cell expansion. The CD140b⁺ cells were then trypsinized and incubated with anti-CD146 microbeads (Miltenyi Biotech) at 4^oC for 15 min. The CD140b⁺CD146⁺ cells were collected and used for single-cell RNA sequencing.

EMSC were obtained by two sequential beadings with magnetic beads coated with anti-CD140b and anti-CD146 antibodies^{17 (link)}. Firstly, the stromal cells were incubated with the 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). The obtained cell suspensions were then loaded onto MS columns (Miltenyi Biotech) with a magnetic field to separate the CD140b⁺ cells. The isolated CD140b⁺ stromal cells were cultured for 7–10 days to allow degradation of the microbeads during cell expansion. The cells were then trypsinized and incubated with the anti-CD146 antibody-coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4 °C to obtain the CD140b⁺CD146⁺ cells for subsequent experiments. The isolated eMSC showed positive expression for CD140b and CD146^{17 (link)}. The stromal cells at passage 1–3 were used in this study.