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Acute Isolation of Cells from Murine Sino-atrial Node.

Bio-protocol (2020-01-05)
Qadeer Aziz, Muriel Nobles, Andrew Tinker
ZUSAMMENFASSUNG

The cardiac conduction system allows the synchronized propagation of electrical activity through heart muscle. This is initiated by the spontaneous activity of the specialized pacemaker cells of the sino-atrial node (SAN). The SAN region underlies automaticity in mammals and therefore has a crucial role in the pathogenesis of cardiac disorders such as arrhythmia. Isolation of SAN tissue and SAN cells is critical to advance our understanding of SAN structure and function in health and disease. Initially, isolation of SAN tissue and SAN cells was carried out in the rabbit owing to its larger size and similar electrical properties to human. This protocol was optimized by Mangoni and Nargeot (2001) for use in mice to take advantage of advancements in transgenic models. Here, we provide a step-by-step guide to dissecting the SAN tissue and isolating pacemaker cardiomyocytes from mouse hearts using an enzyme digestion approach.

MATERIALIEN
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Laminin aus Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Kaliumphosphat, ACS reagent, ≥99.0%
Sigma-Aldrich
Protease aus Streptomyces griseus, Type XIV, ≥3.5 units/mg solid, powder
Sigma-Aldrich
Natriumchlorid, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Albumin aus Rinderserum, lyophilized powder, essentially fatty acid free, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
L-Glutaminsäure Kaliumsalz Monohydrat, ≥99% (HPLC), powder
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D-(+)-Glukose, ACS reagent
Sigma-Aldrich
DL-β-Hydroxybuttersäure Natriumsalz, ~98%
Sigma-Aldrich
Kaliumchlorid, BioXtra, ≥99.0%