Cd31 beads

A)

Zeocin selection

On day 14, differentiating cells were placed under antibiotic selection in endothelial cell media (R&D systems) containing 25 ng/ml of Zeocin (Thermo Fisher Scientific) for 48 h.
OR
B)

CD31 microbead MACS sort

Alternatively, on day 14, differentiating cells can be positively enriched for CD31-positive endothelial cells using CD31 MACS microbeads. Single cell suspensions were prepared using TrypLE (Thermo Fisher Scientific). Cells were pelleted by centrifugation and resuspended in PBS to a final concentration of 1 × 10⁷ cell/ml. Twenty microliters of FCR blocking agent (Miltenyi Biotech) was added per 60 μl of cells as per the manufacturer’s instructions, followed by 20 μl of CD31 beads (Miltenyi Biotech) per 60 μl of cells. Cells were incubated with the CD31 beads at 4 °C for 15 min with gentle shaking. One milliliter of PBS was added to the cell-bead suspension which was then pelleted by centrifugation. The pellet was resuspended in 1 ml of PBS containing 0.04% non-acetylated BSA. Cells were sorted on an autoMACS sorter using the selection program “possel.” The positive fraction was plated into one well of a 6-well plate pre-coated with Matrigel and maintained in endothelial cell media (R&D systems).

Hearts from E15 and E19 embryos, postnatal day 7 (P7) and 14 (P14) pups, and 4-week old mice were used for isolating primary cardiac cells. Briefly, hearts were cut into small pieces and digested with 0.3 mg/mL Trypsin (Invitrogen, Carlsbad, CA, USA), 0.3 mg/mL collagenase II (Worthington, Lakewood, NJ, USA), 5.5 µg/mL DNase I (Sigma, St. Louis, MO) and 1 mg/mL BSA (Sigma) in HBSS solution (Gibco, Waltham, MA, USA) containing 1% penicillin-streptomycin (Gibco). Primary cardiac cells were obtained using Percoll (Sigma) gradient separation. For isolation of Sca-1⁺ CPCs, primary cardiac cells were first stained with CD31 beads (Miltenyi Biotec, Cat# 130-097-418) to deplete CD31⁺ cells using the Magnetic Cell Sorting System (MACS) (Miltenyi Biotec, San Diego, CA, USA), and then the negative portion was stained with Sca-1 beads (Miltenyi Biotec, Cat# 130-098-374) for MACS sorting.

Single-cell suspensions were performed from the intestine by mechanical disruption and passage through filter (Miltenyi Biotec). Lamina propria cells and intraepithelial cells were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to manufacturer’s instructions. In short, tissues were transferred to preheated digestion solution in C tubes (Miltenyi Biotec) and processed by gentleMACS Octo Dissociator (Miltenyi Biotec). The obtained cell suspension was filtered on a 100 μm cell strainer and washed with MACS buffer. Cells were centrifuged (300 × g, 4 °C, 10 min) and counted using the TC20 automated cell counter (Bio-Rad). Leukocytes were depleted or isolated by incubating the cell suspension with CD45 beads (Miltenyi Biotec). For endothelial activation, the CD45-negative lamina propria cells were further isolated using CD31 beads (Miltenyi Biotec).