Sc 271837 af647

Differentiated ventricular-like and pacemaker-like cells were washed with PBS and detached under the use of 1 ml of TrypLE Select 10× (Life TechnoligiesA1217701) for 10 min. The TrypLE reaction was stopped with 1 ml of Medium and cells were centrifuged for 2 min at 200×g. The cell pellet was re-suspended in PBS together with 1:500 Antibodies for connexin 43, connexin 40 and connexin 45 (sc-271837 AF647; sc-374354 AF488; sc-365107 AF594; Santa Cruz). The Antibody was incubated for 15 min at 37 °C. The cells were then centrifuged another time at 200×g for 2 min. The supernatant was removed carefully and the pellet was washed once with PBS. The cells were re-suspended in 500 μL PBS and analyzed with the FACSAria III Cell Sorter (BDBioscences). Data analysis was performed via FACSDiva Software Version 6.1.3 (BDBiosciences). Antibody signals were compared to unstained samples of the same differentiation for background evaluation.

Maturated hiPSC-derived cardiac myocytes with either ventricular-, pacemaker- or conduction system-like specification were detached at day 35 of maturation with 0.5 mL 10 × TrypLE (Thermo Fisher (Waltham, MA, USA)) + 10 μM Y-27632 for 12 min. The digestion was stopped by addition 0.5 mL of the corresponding maturation medium + 10 μM Y-27632. The cells were transferred to a 1.5 mL Eppendorf tube and centrifuged for 3 min at 200× g. the supernatant was removed and the pellet was washed two times with PBS. Then, the pellets were fixed for 10 min with a 3.7% PFA solution. The fixed pellets were washed twice with PBS. The washed pellets were blocked for 1 h with a 0.5% BSA solution. Afterwards, the pellets were washed twice with PBS. The pellets were stained with the following, conjugated antibodies against connexin 43, connexin 40 and connexin 45 (Santa Cruz #sc-271837 AF647; #sc-365107 AF594; #sc-374354 AF488). The respective antibody was used 1:500 in a 0.1% BSA solution for 2 h at RT. The stained pellets were washed twice with PBS and then FACS-analysed.