Characterization of Cardiomyocyte Markers

For flow cytometry and imaging flow cytometry, cells were treated with the FIX and PERM^® Kit (Thermo Fischer Scientific, Waltham, MA, USA) and incubated for 1 h at RT with anti-α-myosin heavy chain (α-MHC; 1:100; Abcam, Cambridge, UK), anti-cardiac troponin T (cTnT, 1:100; Abcam, Cambridge, UK), anti-α-sarcomeric actin (α-SA; 1:100; Abcam, Cambridge, UK), anti-sarcolemmal Ca²⁺ channel (CACNA1C, 1:100; Abcam, Cambridge, UK), and anti-sarcoendoplasmic reticulum Ca²⁺ ATPase protein (Serca2, 1:100; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), followed by incubation with the appropriate secondary antibody conjugated with Alexa Fluor 488, PE-Alexa Fluor 647, or PE-Cy7 (1:200; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [53 (link)]. Cells incubated with isotypes (all from BD) were used as negative controls [54 (link)].
Cytometric analysis was performed with a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) or CytExpert Acquisition and Analysis Software (Beckman Coulter, Brea, CA, USA).