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Acute Isolation of Cells from Murine Sino-atrial Node.

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

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease from Streptomyces griseus, Type XIV, ≥3.5 units/mg solid, powder
Sigma-Aldrich
Potassium chloride, BioXtra, ≥99.0%
Sigma-Aldrich
D-(+)-Glucose, ACS reagent
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Laminin from 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
L-Glutamic acid potassium salt monohydrate, ≥99% (HPLC), powder
Sigma-Aldrich
Potassium phosphate monobasic, ACS reagent, ≥99.0%