ASCs were grown in 100-mm cell dishes (Corning Glass Works, Corning, NY, USA). After reaching 70% to 80% confluence, 5.0 × 105 ASCs were cultured in 5 mL of serum-free low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Scientific, Hemel Hempstead, UK) with or without LPS in low concentration (0.5 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 24 hours. In a variety of experiments including intravenous administration of MSCs, 0.1 mL of 1 × 105 to 1.0 × 106 MSCs has been used for an injection into a mouse [17 (link),30 (link)-32 (link)]. In our protocol, the secretome from 5.0 × 105 ASCs was cultured in 5 mL of DMEM. Therefore, to obtain the equivalent amount (0.1 mL) of secretome, the conditioned media were concentrated 25-fold by using ultrafiltration units with a 3-kDa-molecular-weight cutoff (Amicon Ultra-PL 3; Millipore, Bedford, MA, USA) after stimulation with or without LPS. From here on, LPS-CM and CM refer to the 25-fold concentrated conditioned media which had been obtained from ASCs after stimulation with or without LPS for 24 hours.
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