Pierce cardiomyocyte isolation kit

P0 hearts were dissected in ice-cold 1X PBS. tdTomato fluorescence was used to screen KI-Cre animal hearts from the WT hearts. AVCS was microdissected from KI-Cre hearts (n = 3) guided by tdTomato expression under epifluorescent microscope. Microdissected tissue pieces were pooled from (n = 3) KI-Cre hearts. In order to obtain single cell suspension of microdissected AVCS, we used the Pierce Cardiomyocyte Isolation Kit (Thermoscientific, #88281). The cell yield and viability was determined using hemocytometer counting chambers.

All experimental procedures were performed following institutional and international guidelines and were approved by the local animal welfare authorities (LAGeSo, Berlin, Germany, T-CH0019/20). Timed-pregnant Wistar rat dams were obtained through Janvier Labs (Le Genest-Saint-Isle, France) and housed in individual cages under controlled environmental conditions with ad libitum access to food and water. At embryonic day 18 (E18), adult rats were anesthetized with isoflurane and sacrificed. Pups were segregated from the uterus and transferred into ice-cold phosphate-buffered saline. Hearts were removed and cells were isolated using the Pierce Cardiomyocyte Isolation Kit (Thermo Scientific, Rockford, IL, USA, cat. 88281) following the manufacturer’s instructions. Cells were plated on 35 mm Petri dishes in a density of approximately 2.5x10⁵ cells/cm² and grown in DMEM (Thermo Scientific) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (Bio&SELL) at 37 °C in a 5% CO₂ humidified atmosphere for 7 days. The medium was changed every 2–3 days.