Anti mouse sca 1 pe conjugate

Muscles from hindlimbs from young adult or aged mice were dissected and collected in PBS on ice. Muscles were rinsed with PBS, minced with scissors and incubated in DMEM with Collagenase (0.2%, Biochrom) for 90 min at 37°C and 70 rpm. Digested muscles were washed with PBS/10% FBS, triturated and incubated in Collagenase (0.0125%) and Dispase (0.4%, Life Technologies) for 30 min at 37°C and 100 rpm. The muscle slurry was diluted with PBS/10% FBS, filtered through 100 μm cell strainers and spun down at 500 g for 5 min. Cell pellets were resuspended in FACS buffer (HBSS/2% FBS) and filtered through 40 μm cell strainers and pelleted at 500 g for 5 min. Pellets were resuspended in FACS buffer and stained with anti-mouse CD45 PE conjugate (30-F11, eBioscience), anti-mouse CD11b PE conjugate (M1/70, eBioscience), anti-mouse Sca-1 PE conjugate (D7, BioLegend), anti-mouse CD31 PE/Cy7 conjugate (390, BioLegend) and anti-mouse α7-Integrin Alexa Fluor 647 conjugate (R2F2, AbLab) for 20 min at 4°C on a rotating wheel. Cells were washed with FACS buffer. Live cells were identified as calcein blue positive (1:1,000, Invitrogen) and propidium iodide negative (PI, 1 μg/ml, BD Biosciences). SCs were identified as CD45⁻Sca-1⁻CD11b⁻CD31⁻α7-Integrin⁺. Cell sorting was performed on a FACSAriaIII with Diva Software (BD).