Mice were administered a senolytic cocktail containing 5 mg/kg dasatinib (D-3307, LC Labs, Woburn, MA) and 50 mg/kg quercetin (Q4951, Sigma-Aldrich, St. Louis, MO) in a similar manner as described by Xu et al. [21 (link)] and consistent with a previous study by our laboratory [9 ]. Briefly, 7.5 mg of dasatinib and 75 mg of quercetin were dissolved in 5 mL of 10% polyethylene glycol 400 (PEG 400; 202398, Sigma-Aldrich). We chose this volume of PEG 400 because it allowed us to gavage a 30–45 g mouse with 100–150 μL of senolytic, which is much less than the approximate stomach volume of 400 μL in adult mice [36 (link)]. Senolytics or vehicle (10% PEG) was administered on day 7 and day 10 of the 14-day mechanical overload protocol (Fig. 5a ) using 20-gauge disposable polypropylene feeding tubes (FTP-20–30, Instech, Plymouth Meeting, PA). We chose a hit-and-run approach based on the work from the Kirkland laboratory and these time points because we previously showed that SA β-Gal + cells appear as early as 7 days in a muscle injury model [9 ]. Furthermore, MOV places a constant stress on the muscle such that senescent cells would continue to appear after the initial D + Q gavage and it is unlikely that D + Q kills all of the senescent cells after a single treatment.
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