Collagen 1 coated

Cardiomyocytes were isolated from P1, P3, and P7 pups. Neonatal hearts were dissected, atria were cut off, and ventricles were collected in ice-cold 20 mM 2,3-butanedione 2-monoxime (BDM) solution in HBSS. Ventricles were then cut into 1–2 mm pieces and digested using the gentleMACS dissociation kit (Miltenyi Biotec GmBH, 130-093-235). Cells were then either plated on collagen I–coated (Thermo Fisher Scientific, A1048301) μ-Slides (Ibidi, 80826) or lysed for RNA extraction.

E98 cells were grown on collagen I-coated (10 μg/cm²; Invitrogen) coverslips to 60–80 % confluency over 48 h, and incubated for 1 h with culture medium containing 10 μM EdU (5-ethynyl-2′-deoxyuridine). EdU incorporation was visualized using the click-iT® EdU Imaging kit (Thermo Fisher Scientific, #C10086) via the manufacturers’ instruction. Coverslips were mounted on microscope slides in DAPI-containing Mowiol (Sigma-Aldrich) and images were collected on a Leica DMRA Fluorescence microscope, equipped with a DFC340 FX CCD camera, using 40x and 63x objectives. DAPI- and EdU-positive nuclei were counted automatically using FIJI software [28 (link)].
Migration of E98 cells was assessed in spheroid outgrowth assays as follows. E98 spheroids were generated in hanging drops using methylcellulose (12 mg/mL; Sigma, M6385) in DMEM supplemented with 10 % FCS (2500 cells per spheroid). The next day, individual spheroids were seeded in a 96-well imaging culture dish (BD Falcon, #353219) on top of a confluent mouse astrocyte layer in Matrigel-coated (30 μg/mL PBS; BD Biosciences, #356237) culture wells. 24 h later, cells were fixed and fluorescent (tagRFP) images were collected. Average migration distance of cells from spheroids (n > 37), calculated as change in radius of the spheroid over 24 h, were analyzed semi-automatically using FIJI software.