L3024 5mg

HT-29/kb-seap-25 cells were seeded on 96-well plates at 3 × 10⁴ cells/well and incubated for 24 h at 37°C under 5% CO₂ prior to stimulation (Lakhdari et al., 2010 (link)). Monolayer confluence was checked under the microscope before and after every stimulation. TNFα (1 ng mL^–1; PeproTech), IL-1β (1 ng mL^–1; Invivogen), and LPS from E. coli O111:B4 (1 ng mL^–1; L3024-5MG, Sigma-Aldrich) were used to induce inflammation. The cells were stimulated with the samples (controls and EV preparations) and inflammation inducers for 24 h. The supernatants from all the wells were then revealed with Quanti-Blue^TM reagent (Invivogen) to assess SEAP activity. Cell proliferation was evaluated under all conditions using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS, Promega), according to the manufacturer’s instructions. Absorbance was read at 655 nm for the SEAP activity assay and at 490 nm for the MTS assay using a Xenius (SAFAS Monaco) microplate reader. For surface protein assays, 10⁹ EV ml^–1 were applied directly or after incubation at 37°C for 1 h in the presence or absence of proteinase K (20 μg mL^–1; Qiagen), in order to evaluate a possible role for EV surface proteins in immunomodulation.