Ab187284

TC content in the enriched cultures was assessed by double labeling with phycoerythrin-conjugated monoclonal anti-CD34 (ab187284; Abcam, Cambridge, MA, USA) and rabbit polyclonal anti-PDGFRα (sc-338; Santa Cruz Biotechnology, Dallas, TX, USA). The TCs were analyzed using a BD FACS CantoII cytometer (BD Biosciences, San Jose, CA, USA). Passage-2 cells were washed with D-PBS and harvested by trypsinization. The samples were stained with the above antibodies for 1 h at room temperature. Cells were then incubated with donkey anti-rabbit H&L-labeled secondary antibodies (ab150075; Abcam) for 30 min at room temperature. As a negative control, unstained cell aliquots were incubated with D-PBS under the same conditions. According to the results of the identification, the flow cytometry was adjusted to set the gate and CD34⁺/PDGFRα⁺ cells were sorted to purify the cardiac TCs. The sorted cells were collected in a collecting tube, which was prefilled with complete culture medium and 2% PS, and then centrifuged at 300 × g for 5 min. The cell density was adjusted to 1×10⁵/ml with DMEM/F12 medium containing 10% FBS and 1% PS, following which the cells were seeded into sterile culture dishes and cultured in a humidified atmosphere of 5% CO₂ at 37°C.