title: Optimised protocols for the identification of the murine cardiac side population.
abstract: Cardiac side population (CSP) cells, defined by their ability to efflux the vital dye Hoechst 33342, have been identified as putative cardiac stem cells based on their potential to give rise to both cardiomyocytes and endothelial cells. The CSP phenotype relies on an active metabolic pathway and cell viability to identify a rare population of cells and therefore technical differences in the CSP staining protocol can lead to inconsistent results and discrepancies between studies. Here we describe an established protocol for CSP identification and have optimised a protocol for CSP analysis utilizing an automated cardiac digestion procedure using gentleMACs dissociation and Hoechst 33342 staining followed by dual wavelength flow cytometric analysis.
authors.affiliation: Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle upon Tyne, NE1 3BZ, UK.
authors.forename: Annette
authors.initials: A
authors.lastname: Meeson
authors.forename: Andrew
authors.initials: A
authors.lastname: Fuller
authors.forename: David T
authors.initials: DT
authors.lastname: Breault
authors.forename: W Andrew
authors.initials: WA
authors.lastname: Owens
authors.forename: Gavin D
authors.initials: GD
authors.lastname: Richardson
mesh_terms: D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D003850:Deoxyribonuclease I / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D020784:Matrix Metalloproteinase 8 / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D015203:Reproducibility of Results; D058985:Side-Population Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D001562:Benzimidazoles; D003850:Deoxyribonuclease I; D011402:Pronase; D020784:Matrix Metalloproteinase 8; C017807:bisbenzimide ethoxide trihydrochloride