Biotracker ferroorange live cell dye

iCMs were seeded and allowed to acclimate for 3 days until spontaneously beating was observed. siRNA was used to knockdown CX43 and CX45 for 24 h as described above. iCMs were then washed with PBS. Similar number of HCFs were added to iCM culture to reach confluency. The co-culture was allowed to acclimate overnight. The following morning cells were washed with PBS and treated with FluoroBrite DMEM media containing 25 μM of ferrous ammonium sulfate (Thermo, A15178.36) for 1 h. Following treatment cells were washed with PBS three times and BioTracker FerroOrange Live Cell Dye (MilliporeSigma, SCT210) (1000×) diluted in FluoroBrite DMEM was added to the cells. After 1 h of incubation, cells were washed twice with FluoroBrite DMEM and imaged on a Leica SP8 confocal microscope with live imaging chamber.

iCMs were seeded and allowed to acclimate for 3 days until spontaneously beating was observed. siRNA was used to knockdown CX43 and CX45 for 24 hours. iCMs were then washed with PBS. Similar number of HCFs were added to iCM culture to reach confluency. The co-culture was allowed to acclimate overnight. The following morning cells were washed with PBS and treated with FluoroBrite DMEM media containing 25 μM of FAS (ferrous ammonium sulfate) for 1 hour. Following treatment cells were washed with PBS for three times and BioTracker FerroOrange Live Cell Dye (MilliporeSigma, SCT210) (1000×) diluted in FluoroBrite DMEM was added to the cells. After 1 hour of incubation, cells were washed twice with FluoroBrite DMEM and imaged on a Leica SP8 confocal microscope with live imaging chamber.