Did red fluorescent dye

EVs were isolated from culture medium of neutrophils or hepatocytes by using ExoQuick-TC solution (System Biosciences, Palo Alto, CA) according to the manufacturer's instructions. For EV uptake assay, the EVs isolated from neutrophils were labeled with DiD red-fluorescent dye (Thermo Fisher Scientific, Inc, Waltham, MA) by incubating with 1 mL DiD red-fluorescent dye solution (1:200 dilution) for 15 min. The labeled EV solution was then centrifuged at 100,000 g for 70 min at 4 °C and re-suspended in PBS and rotated at 4 °C for overnight. The EV fractions were incubated with HSCs for 24 hours and subjected to immunofluorescent staining. PBS was used as a negative control. The images were obtained by using LSM 710 confocal microscope (Zeiss, Thornwood, NY).

The DiD-red fluorescent dye (Thermo-Fisher) was used to study distribution of exosomes in vivo. APAP-derived or CON-derived exosomes were labeled with a red fluorescent dye DiD (20 µM final concentration in PBS) by incubation for 30 mins, followed by centrifugation at 10,000 g for 1 h to remove the unbound dye. Exosomes pellets were suspended in PBS and sterilized by passing 0.22 µM filter. To determine cellular distribution by ex vivo imaging analyses, DiD-labeled exosomes from APAP-exposed or control mice were administered into mouse tail vein (C57BL/6J, n = 5/group) via intravenous injection. At 4 h after administration, mouse livers were excised and subjected to confocal microscopy to collect fluorescence from DiD-labeled exosomes.