Figure 3

Measurement of permeability, cytotoxicity, oxidative stress and apoptosis. TsExos (150 μg/mL) were incubated with IPECs for different times, and then, the assays were carried out. A Cell permeability was determined by detecting the fluorescence intensity of FITC-dextran at 493-nm excitation and 517-nm emission wavelengths. B Cell cytotoxicity determined by assessing the level of LDH at OD₄₉₀. Cellular oxidative stress was detected by a fluorescence microplate reader at 488-nm excitation and 525-nm emission wavelengths (C) and a fluorescence microscope with a magnification of 20× PL FL and a scale bar of 100 μm (D). Apoptosis of IPECs was determined by staining with annexin V and PI followed by flow cytometry (E), by staining with Hoechst 33258 (F) and by analysing the relative expression of Bax and Bcl-2 using Western blotting (G). All assays were performed in triplicate, and the data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the IPEC + PBS group.