Sc 133256

A volume of 100 μl of PBS containing Cy5.5-labeled Akk OMVs (0.1 mg/ml) was incubated with Caco-2 and HT-29-MTX-E12 cells for 24 hours at 37°C, respectively. The cells were washed three times with PBS and stained with Hoechst before visualizing uptake of OMVs under CLSM, respectively. Similarly, a volume of 100 μl of PBS containing Akk OMVs (0.1 mg/ml) was cultured with Caco-2 cells along with LPS (5 μg/ml) for 24 hours at 37°C. Then, the cells were processed for immunofluorescence staining, including occludin (sc-133256, Santa Cruz Biotechnology) and ZO-1 (AF5145, Affinity Biosciences), and then stained with Hoechst before CLSM observation. A volume of 100 μl of PBS containing Akk OMVs (0.1 mg/ml) was cultured with HT-29-MTX-E12 cells for 24 hours at 37°C. Then, the cells were processed for Alcian blue staining (C0155S, Beyotime Biotechnology).

The proximal colons were extracted from gut disorder mice 5 days after oral administration of Akk OMV suspension containing 20 μg of proteins by gavage. To analyze mucus barrier, the samples were processed for Alcian blue staining (C0155S, Beyotime Biotechnology) in accordance with the manufacturer’s instructions. Fiji (ImageJ) was used to quantitatively analyze the area of stained cells and the percentage of goblet cells in the intestinal epithelium. To analyze physical barrier, the samples were processed for immunofluorescence staining in accordance with the manufacturer’s instructions, including occludin (sc-133256, Santa Cruz Biotechnology) and ZO-1 (AF5145, Affinity Biosciences). Levels of cytokines including IL-10 (EK210/4, MultiSciences), IL-13 (EK213/2, MultiSciences), and ACE2 (EK1188, Boster Biological Technology) in colon tissue as well as IFN-γ (EK280/3, MultiSciences) and CRP (EK294/2, MultiSciences) in serum were measured by commercially available ELISA kits. To observe uptake of Akk OMVs by intestinal epithelial cells, a solution of Cy5.5-labeled OMVs (0.1 mg/ml) was injected into the intestinal segment tied at both ends and the treated mice were euthanized for collecting intestinal tissues 2 hours later. Then, the tissues were processed for CLSM observation.