Freezone 2.5 plus freeze dry system

At 24 h after EV treatment, BMDMs were washed with PBS twice and lysed in 80% methanol containing 50 μM (+)-10-camphorsulfonic acid, 400 μM L-methionine sulfone, and 400 μM piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) as internal standards. The cells were incubated for 15 min at -80°, then scraped and centrifuged at 14,000 g for 5 min at 4°. The supernatant was collected and filtered using a Millipore 5 kDa cut-off membrane to remove solubilized proteins. The dried metabolites were dissolved in Milli-Q water after evaporation of the aqueous-layer extracts under vacuum using a FreeZone 2.5 Plus freeze dry system (Labconco, Kansas City, MO). The concentrations of intracellular metabolites were analyzed with a CE/MS (CE, Agilent G7100; MS, Agilent G6224AA LC/MSD TOF; Agilent Technologies, Palo Alto, CA) controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies), as described previously (18 (link)). The same validation was performed on BMDMs stimulated with LPS for 1.5 h at 24 h after EV treatment. For metabolite analysis on myotube-derived EVs, EVs extracted as above were used. The relative abundance of each metabolite to the control group or LPS group was calculated. Quadruplicate cell cultures were analyzed for each condition tested.

Eighty percent MeOH [water containing 50 μM ( +)-10-camphorsulfonic acid, 400 μM L-methionine sulfone, and 400 μM piperazine-1,4-bis (2-ethanesulfonic acid)] was added to each dish and incubated for 15 min at − 80 °C. Cells were scraped and centrifuged at 14,000 × g for 5 min, and the supernatant was centrifuged at 14,000 × g for 90 min at 4 °C using a 5 kDa cut-off membrane (Merck Millipore, MA, USA) to remove the solubilized protein. The dried metabolites concentrated by evaporation of the aqueous layer extracts with a FreeZone 2.5 Plus freeze-dry system (Labconco, Kansas City, MO) were dissolved in Milli-Q water. The intracellular metabolites were analyzed using a capillary electrophoresis-mass spectrometry (CE/MS, Agilent G7100; MS, Agilent G6224AA LC/MSD TOF; Agilent Technologies, Palo Alto, CA) controlled by the MassHunter Workstation Data Acquisition software (Agilent Technologies)^{33 (link)}.