Lb agar medium

The bacterial translocation assay was carried out as described (Kobayashi et al., 2019 (link)). Briefly, bacterial cells obtained from the culture in LB were suspended in Hanks’ balanced salt solution (HBSS) containing 15 mM HEPES (pH 7.4). The suspended bacterial cells (2 x 10⁵ cells) were added at a multiplicity of infection (MOI) of 5 to the apical side of T84 cells grown on Transwell filters (pore size 3.0 µm). In this procedure, the number of bacteria in each bacterial suspension was confirmed by the plating method.
To assess the extent of bacterial translocation from the apical side to the basal side by increasing the number of bacteria present in the lower chamber (the basal side) as much as possible, we performed this experiment for the maximum period (9 hr) during which the T84 intestinal epithelial monolayer did not detach from the Transwell. After infection at 37°C with 5% CO₂ for 9 hr, the changes in TER were recorded and the number of bacteria present in the culture medium (10 μL) of the lower chamber was calculated by the plating method using LB agar medium (BD Biosciences).

Sanguinarine chloride was purchased from MedChamExpress (Monmouth Junction, NJ, USA), and polymyxin B was purchased from Inalco (Tuscany, Italy). The natural product library was obtained from MedChamExpress (Monmouth Junction, NJ, USA). Luria–Bertani (LB) medium and LB agar medium were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Mouse IL-6/IL-1β/TNF-α ELISA kits for the detection of cytokines were purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China). ATP and AKP kits were purchased from Beyotime Biotechnology (Shanghai, China). BacLight Bacterial Membrane Potential Kits were obtained from Invitrogen (Carlsbad, CA, USA). TO-PRO-3 dye was purchased from AAT Bioquest (Pleasanton, CA, USA).