Figure 3.

ONV Effects on Caco-2/HT29-MTX Intestinal Barrier

(A) Representative images of IBs obtained with identical image acquisition parameters for AdipoRed and Hoechst 33258 on a Cytation 3 platform, showing the decrease in TG content post-treatment with ONV (3 h, 1 μg/mL) (objective ×4, IBs grown in 96-well inserts; objective ×20, IBs grown in six-well inserts). (B) TG levels in IBs 24 h post-treatment with ONVs, measured by quantification of AdipoRed fluorescence intensities in IB cells sorted by flow cytometry (n = 3). (C) Volume and number of lipid droplets in 3T3-L1 MBX differentiated adipocytes co-cultured with ONV-pretreated IBs (n = 6–12 replicates). (D) Representative images of 3T3-L1 MBX differentiated adipocytes in 96-well E-plates after 24 h of co-culture with IBs. Contrast phase (left panels) and corresponding merged AdipoRed-Hoechst 33258 (right panels) are shown. (E) TGs in adipocytes. Data are mean ratios of fluorescence intensities (AdipoRed-Hoescht 33258, n = 12 replicates) quantified with a Cytation 3 cell imaging reader. (F) xCELLigence analyses of 3T3-L1 MBX differentiated adipocytes, co-cultured with ONV-pretreated IBs during 24 h. The data represent real-time monitoring of 3T3-L1 MBX cell index normalized to the cell index at T = 0 (mean delta cell index, every 5 min during 50 cycles, then every 15 min) for 22 h. The decrease of cell index in the 3T3-L1 MBX + pre-treated IBs versus 3T3-L1 MBX + control IBs indicated a decrease in adipocyte adherence due to lipid accumulation.²⁸ *p < 0.05, student t test.