Il 21

Culture supernatants were collected at 24 h from duplicate wells seeded at 50,000 cells/well in a 24-well plate, with 0.5 mL total medium volume per well. Controls were prepared from the wells receiving complete medium only, without EVs. RayBio^® C-Series Canine Cytokine Array Kit 1, which can detect a total of 40 individual canine cytokines, was purchased from RayBiotech (RayBiotech, Peachtree Corners, GA, USA) and utilized according to manufacturer instructions. Briefly, after blocking, samples were placed in each well overnight at 4℃. The next day, samples were twice washed, and HRP-streptavidin incubation was performed overnight at 4℃ before a third wash. The next day, chemiluminescence detection was used to recognize the antibody levels expressed in the membrane. For comparison of their expression levels, internal positive controls were used to define the signal strength. Two duplicate spots were used for all samples. Data were analyzed in Microsoft® Excel-based Analysis Software Tools (RayBiotech) for each array kit via automatic analysis. The canine IFN-r (R&D Systems), IL-21, TNF- $\alpha ,$ and Rantes quantitative ELISA kits (RayBiotech) were applied to analyze these concentrations in the supernatant under the exposure with or w/o EVs.

ELISAs of serum, BALF, and lung-tissue homogenates were conducted using commercial kits for IgE (Crystal Chem, Chicago, IL, United States), IL-6 and IL-17A (Anogen, Mississauga, Canada), IL-21, p-STAT3 (Tyr705), and total STAT3 (RayBiotech, Peachtree Corners, GA, United States), HDAC2 (EpiGentek, Farmingdale, NY, United States), and hypoxia inducible factor (HIF)-1α (Cell Biolabs, San Diego, CA, United States) according to the manufacturer’s instructions.

Colon tissue was homogenized in 1 mL PBS and centrifuged (13,000 rpm for 20 min at 4 °C). Supernatant was filtered (0.22 μm), and protein concentration was determined by Bradford assay. The presence of cytokines was analyzed in supernatant fractions with mouse antibody arrays Q1 (ABIN625794; RayBiotech) following the manufacturer’s instructions. ELISA kits were used to quantify IL-22, IL-13, IL-10, TGF-β, IL-23, IL-21, IL-6, and TNF-α (RayBiotech).