5 5 dithiobis 2 nitrobenzoic acid dtnb d8130

Acetylcholinesterase (AChE, C3389), acetylthiocholine iodide (ATCh, 01480), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, D8130), and Tris-HCl buffer (1185-53-1) were purchased from Sigma-Aldrich (Saint Louis, MO, USA/Poznań, Poland). Folin–Ciocalteu reagent (F-C, 694350111) and other analytical grade reagents were purchased from Avantor (formerly P.O.Ch. Gliwice, Poland).

Electron microscopy was done in the core imaging facility (Department of Anatomical Sciences and Neurobiology, University of Louisville) using the Phillips CM12 electron microscope with digital cameras. Briefly, the heart tissues were fixed in 3% glutaraldehyde and cut into ultrathin sections at 60–90 nm thick and collected onto grids. The grids were stained with uranyl acetate for negative staining. The exosomes were confirmed by AChE activity (based on the Ellman assay) 26 (link). Briefly, 2 μl of exosomes were resuspended in PBS diluted in 298 μl of the AChE assay working solution [1.25 mM Acetylthiocholine (A5751; Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB, D8130; Sigma- Aldrich) in 0.1 M phosphate buffer at pH 8.0] and incubated at 37°C in the dark for 30 min. Optical density (OD) was measured at 412 nm to quantify AChE activity in the exosome solution.