Immunofluorescent Characterization of iPSC-Derived Cardiomyocytes

IPSc-derived cardiomyocytes were cultured on glass slides coated with Synthemax II-SC (Corning) in RPMI-1640/B-27 with Y27632 (5 μM) media for 2 days. Cells were fixed with 4% formaldehyde for 15 min at room temperature. Fixed cells were blocked in antibody buffer (5% BSA, 0.1% Tween-20 in PBS) for 1 h at room temperature. Following blocking, cells were incubated overnight at 4 °C with cardiac troponin-T antibody (abcam, ab45932, 1:200), α-Actinin antibody (Sigma, A7811, 1:800), and/or Connexin 43 (Cell Signaling Technology, 1:100) in antibody buffer. After the overnight incubation, cells were washed three times in antibody buffer. Following washing, cells were incubated with Alexa-Fluor conjugated secondary antibodies (Thermo Fisher Scientific, Alexa 488 Donkey anti-Rabbit IgG and Alexa 594 Goat anti-Ms IgG1) at a 1:1,000 dilution in antibody buffer for 1 h at room temperature. Nuclei were stained by DAPI at 1 μg/ml and Wheat Germ Agglutinin (Thermo Fisher Scientific, W21404) was used to stain cell membrane for 10 min at room temperature in antibody buffer. Following washing in PBS to remove unbound complexes, sarcomeres were analyzed using a Zeiss LSM510 confocal microscope. Images were processed with Zeiss software (Axiovision Rel4.8 and Zen Blue). Circularity measurements were made by comparing cardiomyocyte length to width and were expressed as a circularity index whereby circularity index = width/length^{15 (link)}. Sarcomere distance measurements were made with ImageJ^{15 (link)}. All measurements were made with a double-blinding method.