Protein quantitation assay kit

Mice were euthanized and the intestine removed. The luminal contents of the intestine were removed by gently flushing the intestine with 10 ml of ice-cold PBS. The intestine was then opened longitudinally, the mucus collected by mild physical separation using round forceps, soaked in PBS, then agitated on a rotator at 70 rpm/min for 15 min and centrifuged at 500 × g for 15 minutes at 4°C. The supernatant was then followed by differential centrifugation and sucrose gradient centrifugation to isolate and purify the exosomes using a previously described protocol^{38 (link)}. Protein concentration was determined using the Bio-Rad Protein Quantitation Assay kit with bovine serum albumin as a standard. Each lot of IDENs used for this study was also FACS analyzed for CD63, an exosomal marker, and A33, a marker of intestinal epithelial cells. Exosomes used in this study were always more than 90% of CD63⁺ A33⁺IDENs.

To prepare plant exosome-like nanoparticles (ELNs), peeled Hawaiian ginger roots (Simply ginger, PLU#:4612), carrot, garlic, turmeric roots and grapefruit were used for isolation and purification of ELNs using a previously described method (Mu et al., 2014 (link)). Briefly, the plants listed above were peeled and then homogenized in a high-speed blender for 1 min. The juice was collected after net filtration. The supernatant was collected after centrifugation at 1,000x g for 10 min, 2,000x g for 20 min, 4,000x g for 30 min, and 10,000x g for 1 h. The pellets containing nanoparticles derived from each plant were spun down at 100,000x g for 1.5 h at 4°C. The isolated exosomes were further purified in a sucrose gradient (8, 30, 45, and 60% sucrose in 20 mM Tri-Cl, pH 7.2), followed by centrifugation at 100,000x g for 1.5 h at 4°C. Purified GELNs were fixed in 2% paraformaldehyde and imaged under a Zeiss EM 900 electron microscope using a previously described method (Mu et al., 2014 (link)).
The purity of GELNs was evaluated by calculating the ratio of particle to protein (Webber and Clayton, 2013 (link)). The size distribution of GELNs was analyzed using a Zetasizer Nano ZS (Malvern Instrument, UK) at a flow rate of 0.03 ml per min. The protein concentration of GELNs was determined using a BioRad Protein Quantitation Assay kit with bovine serum albumin as the standard.

Isolation of gut EVs was performed as described previously (Deng et al., 2015 (link)). Briefly, mice were euthanized and the intestine removed. The luminal contents of the intestine were removed by gently flushing the intestine with ice-cold PBS. The intestine was then opened longitudinally and gently washed with ice-cold PBS. The mucous was scraped off by mild physical separation using a glass slide, soaked in washing PBS, agitated on a rotator at 70 r.p.m. min⁻¹ for 15 min and centrifuged at 500×g for 15 min at 4°C. The supernatant was then followed by differential centrifugation: 4000 rpm for 30 min, 8000 rpm for 60 min, 36000 rpm for 90 min. The pellets from the 8000 rpm or 36000 rpm centrifugations were used for microparticles and exosome-like particles, respectively. Protein concentration was determined using the Bio-Rad Protein Quantitation Assay kit with BSA as a standard.