The hiPSCs, hCVPCs and hCMs analyzed by flow cytometry as previously described [32 (link),33 (link),37 (link)] and incubated with antibodies: PE-conjugated SSEA4 (eBioscience, 12-8843-42, 1:100), PE-conjugated SSEA1 (eBioscience, 12-8813-42, 1:50), or un-conjugated cTnT (Abcam, ab8295, 1:200) and ki67 (Abcam, ab16667, 1:200) with PE-conjugated secondary antibody (eBioscience, 12-4010-87, 1:200) or isotype-matched negative controls. Cells were then analyzed by FACS (FACS tar Plus Flow Cytometer, Becton-Dickinson, San Jose, CA, USA).
Cell sorting was performed using a FACS sorter (Beckman Moflo Astrios) based on the SSEA1 detection as we reported previously [37 (link)]. Briefly, The Mix cells were dissociated 0.05% Trypsin-EDTA (Thermo Fisher Scientific) for 5 min at 37 °C and washed once with the wash buffer (1% FBS in Dulbecco's phosphate-buffered saline, DPBS, Invitrogen, 14190144) after collected. The cells were then incubated with the primary antibody (PE-conjugated SSEA1, eBioscience, 12-8813-42, 1:50) for 1 h at 4 °C after blocking. Then the suspensions were filtered with a 40 μm cell strainer and sorted using Beckman Moflo flow cytometer. The SSEA1+ cells (Mix-hCVPCs) and SSEA1- cells (Mix-hCMs) were collected and used for further analysis.
Cell sorting was performed using a FACS sorter (Beckman Moflo Astrios) based on the SSEA1 detection as we reported previously [37 (link)]. Briefly, The Mix cells were dissociated 0.05% Trypsin-EDTA (Thermo Fisher Scientific) for 5 min at 37 °C and washed once with the wash buffer (1% FBS in Dulbecco's phosphate-buffered saline, DPBS, Invitrogen, 14190144) after collected. The cells were then incubated with the primary antibody (PE-conjugated SSEA1, eBioscience, 12-8813-42, 1:50) for 1 h at 4 °C after blocking. Then the suspensions were filtered with a 40 μm cell strainer and sorted using Beckman Moflo flow cytometer. The SSEA1+ cells (Mix-hCVPCs) and SSEA1- cells (Mix-hCMs) were collected and used for further analysis.