Isolation and Analysis of Cardiac Side Population Cells

CSPs were isolated as previously described [21 (link)]. The minced cardiac tissue from freshly isolated mouse hearts was digested using collagenase type 2 (LS004174, Worthington Biochemical Corp, Lakewood, NJ, USA) and dispase (17105–041, Life Technologies). The cells were resuspended at a density of 1.0 × 10⁶/mL in PBS containing 3% fetal bovine serum. The cells were incubated in 1 μg/mL Hoechst 33342 dye (H3570, Molecular Probes) for 60 min at 37°C in the dark, with or without 50 μM verapamil. Following incubation, the cells were analyzed using Altra flow cytometric analysis (Beckman Coulter, Fullerton, CA, USA). Hoechst 33342 dye was excited at 350 nm using an ultraviolet laser. Fluorescent emission was detected using 450-nm bandpass (Hoechst blue) and 675-nm longpass (Hoechst red) filters, respectively. Isolated CSPs were used for in vitro incubation experiments or directly centrifuged onto slides using a CytoSpin Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 71.8 × g for 5 min. For the β-galactosidase expression analysis, isolated CSPs and residual cardiomyocytes were fixed with 0.25% glutaraldehyde for 10 min and stained in X-gal staining solution for 1 h. For normal immunofluorescence staining, CSPs were fixed with 4% paraformaldehyde for 10 min and stained using the method described for fresh-frozen section staining.