Sprague dawley neonatal rats

NRVMs were isolated using collagenase digestion and adhesion differential from fibroblasts as described.[10 (link)] Briefly, Sprague Dawley neonatal rats (1-2 days old) (Taconic) were decapitated and the hearts were isolated. Following removal of the atria, the ventricles were cut into small pieces and digested first in .05% trypsin/EDTA (Corning) overnight, then in collagenase II (Gibco) for 30 minutes. Cells were then spun at 100 × g followed by a 40 minute pre-plating process on non-treated plates to allow the fibroblasts to adhere. The non-adherent NRVMs were then transferred to cell culture-treated dishes in MEM alpha media (Gibco) with 10% FBS.
The study was performed under protocol #13-08-29-01, which has been approved by the University of Cincinnati Institutional Animal Care and Use Committee, and the animals received humane care in compliance with the National Research Council's criteria as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health.

Primary CMs and aCCs from the ventricles, and primary postganglionic sympathetic neurons from the SCG were isolated from 2 d old (p2) Sprague–Dawley neonatal rats (Taconic Biosciences) using established protocols approved by Northeastern University’s Institutional Animal Care and Use Committee (19–0104R).^{[17 (link),20 ]} In brief, rat pups were euthanized by decapitation and isolated tissue kept on ice in Hibernate-A (BrainBits) while tissue from every pup was collected. Once all tissue was harvested, SCGs were enzymatically dissociated sequentially in collagenase I (305 units mg⁻¹ in Hank’s Balanced Salt Solution (HBSS), Gibco) for 60 min and then in 0.5x Trypsin in HBSS for 15 min. Partially dissolved ganglia was then further broken up via mechanical trituration with a fire polished pipette. Dissociated sympathetic neurons were counted, then cryopreserved with 10% dimethyl sulfoxide (Fisher) at 1 × 10⁶ cells mL⁻¹ for use on-demand. The following day, cardiac tissue was dissociated by serial collagenase II (305 units mg⁻¹ in HBSS, Gibco) digestions at 37 °C after an overnight incubation in 0.5% v/v trypsin in HBSS overnight at 4 °C. CMs were purified from aCCs via differential attachment in which any unattached cells after 1 h were considered enriched CMs. CMs and aCCs were counted and seeded within 1–2 h following enrichment.