EVs isolated from conditioned medium of C2C12 cells were labeled labeled with the membrane dye DiR (Xenolight, Perkin Elmer) following manufacturers recommendations. DiR is a near-infrared lipophilic dye that binds the lipid bilayer surrounding EVs [20 (link)]. Its emission spectra is only visible using infrared CCD imaging. We injected 100 µl of DiR dye alone or DiR-labeled EVs at 1.0 mg/ml and then imaged (Ex =710, Em=790) 24 hrs later using an Ami X spectral imaging instrument. Bone marrow cells were flushed from long bones of young adult mice and adherent and non-adherent cells cultured and treated (50µg/ml EVs) with normal C2C12 EVs and miR-34a overexpressed EVs for 18 hrs. The cell lysate was prepared for western blot analysis as per our published method [57 (link)]. In brief, protein was extracted from cell culture lysate, subjected to SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were incubated with a polyclonal antibody against Sirt1 (Millipore Anti-Sirt1 Cat # 07-131), and beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 ºC, followed by incubation with appropriate secondary antibody. Proteins were visualized with an ECL Western blot detection system (Thermo Scientific, Waltham, MA).
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