Anti cd31 and cd34 microbeads

Human and mouse PCs were immunosorted as CD31^–CD34⁺ cells from human and mouse myocardial samples, as previously described (13 (link)). Briefly, samples were finely minced using scissors and a scalpel until nearly homogeneous and digested with Liberase (Roche) for up to 1 hour at 37°C, with gentle rotation. The digest was passed through 70-, 40-, and 30-μm strainers. Finally, the cells were recovered and sorted using anti-CD31 and -CD34 microbeads (Miltenyi Biotec) to deplete the population of CD31⁺ ECs and select CD31^–CD34⁺ cells. Cells were expanded in Endothelial Cell Growth Medium 2 (ECGM2, PromoCell) employing human or mouse recombinant GFs, and used for experiments between passage 4 and 7.

Cardiac PCs were immunosorted as CD31neg/CD34pos cells from human myocardial samples, and expanded in a dedicated medium supplemented with human recombinant growth factors and 2% v/v foetal calf serum (FCS) (ECGM2 complete kit, C-22111, PromoCell) as previously described [11 (link),28 (link)]. Briefly, samples were finely minced using scissors and scalpel until nearly homogenous and digested with Liberase (Roche) for up to 1 h at 37 C, with gentle rotation. The digest was passed through 70-, 40-, and 30-μm strainers. Finally, the cells were recovered and sorted using anti-CD31 and -CD34 microbeads (Miltenyi) to deplete the population of CD31pos ECs and select CD31neg/CD34pos cells, which distinguish a population of perivascular cells in situ [11 (link),28 (link)]. After expansion to passage 3, the purity of the cell population was verified using immunocytochemistry (ICC) or flow cytometry [11 (link),28 (link)].
Human coronary artery ECs (CAECs) were purchased from PromoCell and expanded in the same medium used for PCs. All cells used in the present study tested negative for mycoplasma contamination (assessed using the PCR Mycoplasma Test Kit I/C, PromoCell, cat# PK-CA91-1096). Cells were used between passages 4 and 7.