Cardiac Cell Population Isolation

Mice were sacrificed under deep isoflurane anesthesia and the hearts were rapidly cannulated. Hearts were retrogradely perfused with a liberase/trypsin solution as described above (17 (link)). The resulting single-cell suspension was centrifuged at 20g for 10 seconds to pellet rod-shaped cardiomyocytes (43 (link)) and passed through a 40 μm cell strainer. The supernatant containing the nonmyocyte fraction was incubated sequentially for 15 minutes with biotin-labeled anti-CD31 (catalog number 130-111-539, Miltenyi Biotec; for endothelial cells), anti–PDGFR-β (130-1089-866, Miltenyi Biotec; for pericytes), and anti–PDGFR-α antibodies (130.101.1905, Miltenyi Biotec; for fibroblasts) followed by 15 minutes’ incubation with anti-biotin magnetic microbeads (130-105-637, Miltenyi Biotec). Cells were separated from the cell suspension by magnetic-assisted sorting employing MS columns (130-042-201, Miltenyi Biotec) according to the manufacturer’s protocol (43 (link)).

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Michel K., Herwig M., Werner F., Špiranec Spes K., Abeßer M., Schuh K., Dabral S., Mügge A., Baba H.A., Skryabin B.V., Hamdani N, & Kuhn M. (2020). C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness. JCI Insight, 5(22), e139910.

Publication 2020

anti-biotin magnetic microbeads

Anesthesia Anti antibodies Biotin Cardiomyocytes Cell Endothelial cells Fibroblasts Hearts Isoflurane Liberase Mice Microbeads Pdgfr β Pericytes Seconds Trypsin

Corresponding Organization : Ruhr University Bochum

Other organizations : University of Duisburg-Essen, University of Münster

Top 5 similar protocols

Variable analysis

independent variables

Isoflurane anesthesia
Enzymatic digestion with liberase/trypsin solution
Mechanical separation of cell types using cell strainer and magnetic-assisted sorting

dependent variables

Isolation of single-cell suspension containing different cell types (cardiomyocytes, endothelial cells, pericytes, fibroblasts)

control variables

Perfusion pressure and flow rate during heart cannulation and retrograde perfusion
Centrifugation speed and duration for pelleting rod-shaped cardiomyocytes
Incubation time with antibodies and magnetic beads during cell separation

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