Imaging Neonatal Mouse Cardiomyocytes

Neonatal mouse cardiomyocytes were isolated from the ventricles of 3-day-old mice homozygous for PLN R14del or wild-type C57Bl6/J littermates after decapitation. After trypsinization, cells were passed through a 40μm cell strainer and plated for 70 min to get rid of large debris and fibroblasts. Cells were plated on culture plates with microcontact printed flexible PDMS substrates at 50.000 cells per well and cultured at 37 °C under 5% CO₂. Media for the first 24 h was MEM supplemented with 5% FCS and 1% penicillin–streptomycin, hereafter DMEM with 10% FCS and 1% penicillin–streptomycin was used. Media was additionally supplemented with 1% BrdU for the first 4 days. Cells where treated with either 15 μM PLN-ASO or PBS for 30 h. After an additional 24 h, cells were incubated with 2.5 μM Fluo-4 (F14201, Invitrogen) for 20 min at RT, followed three washes with warm PBS and placed in RPMI media with HEPES 1:1000. Cell were then paced at 3 Hz and live imaging was recorded at 74 fps using a DeltaVision Microscope System from GE Healthcare^{52 (link)}.