Zeocin

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).

Retrovirus was produced by transfecting the constructed retroviral vectors (CD148 WT, Q276P/R326Q) into Phoenix packaging cells as described previously.¹⁵ The empty vector was used to produce control virus. In brief, Phonix cell transfection was performed using the calcium phosphate‐DNA coprecipitation method in the presence of chloroquine. About 60 h post transfection, viral particles were collected, passed through a 0.45 μM filter, then concentrated using Retro‐X™ Concentrator (Clontech, Mountain View, CA). The concentrated retroviruses were added to A431D cells, and stable cells were selected with 400 μg/ml Zeocin (Invitrogen, Carlsbad, CA). The Zeocin‐resistant cells were stained with a PE‐conjugated anti‐CD148 antibody (R&D Systems), and the stable cells that express comparable levels of CD148 were sorted using a BD FACSAria II flow cytometer (BD Biosciences) as described.¹⁵ Control A431D cells were generated by the infection of retrovirus that was produced with the empty retroviral vector.