Hoechst 33342 dye

Side population (SP) cells were isolated as described previously using Hoechst 33342 dye (Lonza, Basel, Switzerland) with some modifications [19 (link), 20 (link)]. Briefly, cells were resuspended at 1 × 10⁶/mL in pre-warmed DMEM supplemented with 5% FBS. Hoechst 33342 dye was added at a final concentration of 2.5 μg/mL in the presence or absence of verapamil (75 μM; Sigma-Aldrich) and the cells were incubated at 37°C for 60 min or 90 min with intermittent shaking. Analyses and sorting were performed with a FACSAria II cell sorter (Becton Dickinson). The Hoechst33342 dye was excited at 357 nm and its fluorescence was analyzed using dual wave-lengths (blue, 402–446 nm; red, 650–670 nm).

Cultured fibers were fixed with 4% paraformaldehyde in saline for 10 min at room temperature, washed with saline solution, and pre-treated with blocking buffer [5% BSA in PBST (PBS with 0.1% Triton X-100)] at 4°C overnight. The cells were immunostained with primary antibodies (S1 Table) in the antibody buffer [1% BSA in PBST] overnight at 4°C and incubated with secondary antibodies in the antibody buffer for 1 h. After being mounted within a SlowFade^TM antifade mountant (Thermo Fisher) containing Hoechst 33342 dye (Lonza), the samples were observed using a confocal laser fluorescent microscope (LSM780, Carl Zeiss).

Side population (SP) cells were isolated as described previously using Hoechst 33342 dye (Lonza, Basel, Switzerland) with some modifications [4 (link), 15 (link)]. Briefly, cells were resuspended at 1 x 10⁶/mL in pre-warmed DMEM supplemented with 5% FBS. Hoechst 33342 dye was added at a final concentration of 2.5 μg/mL in the presence or absence of verapamil (75 μM; Sigma-Aldrich) and the cells were incubated at 37°C for 60 min or 90 min with intermittent shaking. Analyses and sorting were performed with a FACSAria II cell sorter (Becton Dickinson). The Hoechst33342 dye was excited at 357 nm and its fluorescence was analyzed using dual wave lengths (blue, 402–446 nm; red, 650–670 nm).