L 80 ultracentrifuge

HeLa or melan‐Ink cells were harvested, washed with ice‐cold PBS and then homogenized in lysis buffer (0.25 M sucrose, 10 mM HEPES pH 7.4, 1 mM EDTA and 1× protease inhibitor cocktail) at 4°C using Dounce homogenizer. Cell lysate was clarified by centrifugation at 700 g at 4°C for 10 min. Further, homogenates were fractionated on sucrose step gradient (top to bottom: 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 M sucrose buffers layered manually) by ultracentrifugation (Beckman L‐80 ultracentrifuge) at 120,000 g for 1 h at 4°C in SW60Ti rotor (Beckman). Fractions were collected from top to bottom and subjected to immunoblotting.

In some experiments, C-DGUC was performed as described previously with some modifications [34 (link),55 (link)]. In brief, gut-lavage fluids were pooled from sham or CLP mice (10 mice in each group) and concentrated into a final volume of 30 mL using the Amicon ultra-15 centrifugal filter unit (Merck) per the manufacturer’s instructions. The concentrated samples were placed on 0.8 M and 2 M sucrose cushions and centrifuged at 100,000× g for 120 min at 4 °C in as SW32Ti rotor using the L-80 ultracentrifuge (Beckman Coulter). The interface layer was harvested, diluted five-fold with HEPES buffered saline (HBS), filtered through the 0.22-μm filter unit (Merck) and then repeated for sucrose ultracentrifugation in a SW41Ti rotor as described above. The interface layer was harvested and diluted in 2.3 mL of HBS and mixed with an equal volume of 60% iodixanol to achieve a 30% iodixanol concentration containing EVs. This solution was overlaid with 20% and 5% iodixanol solutions and centrifuged at 200,000× g for 120 min at 4 °C in an SW32Ti rotor. Ten consecutive 1-mL fractions were diluted in PBS and centrifuged at 100,000× g for 120 min to pellet the EVs. Each pellet was resuspended in 100 µL of PBS and the protein concentration was measured as described above.

Subcellular fractionation was carried out using a protocol described previously (Mahanty et al., 2016 (link)). Briefly, melanocytes were harvested, washed with 1× PBS and suspended in 0.25 M sucrose buffer (0.25 M sucrose, 1 mM EDTA, 25 mM HEPES pH 7.4, 0.02% sodium azide and protease inhibitor cocktail). Cells were homogenized on ice using a Dounce homogenizer and then clarified by centrifugation at 600 g for 10 min at 4°C. The cell lysate was fractionated on a sucrose step gradient (2.0 M, 1.6 M, 1.4 M and 1.2 M sucrose buffers manually layered from bottom to top in an ultracentrifuge tube) using a SW55Ti rotor by spinning at 160,000 g at 4⁰C for 4–6 h in a Beckman L-80 ultracentrifuge. Fractions were manually separated and subjected to immunoblotting. In Fig. S4, the percentage enrichment of each protein in the fraction was calculated from the protein band densities, normalized to that in the first fraction and then plotted as a graph.