Fig. 2.

Optimization of the apical VDC medium for co-incubation of EPEC, L. reuteri or R. gnavus and human T84 cell epithelia. (A) Growth of EPEC, L. reuteri (LR) and R. gnavus (RG) in LB, MRS and BHI-YH broth. Bacteria were incubated in the VDC without host cells and OD₆₀₀ was monitored over 5 h (EPEC and L. reuteri) or 24 h (R. gnavus). Mean±s.d., n=4. (B) Influence of BHI-YH medium on intestinal epithelial integrity and barrier function. Differentiated T84 cells were incubated in the VDC for 22 h with BHI-YH (BHI), DMEM/F-12 (F-12) or a 1:1 mixture of both media (BHI/F-12) on the apical side and DMEM/F-12 on the basal side. Epithelia were stained for F-actin (green) and occludin (red). Scale bars: 10 µm. Images are representative of n=6. (C) Growth of EPEC, L. reuteri and R. gnavus in BHI-YH (BHI), DMEM/F-12 (F-12) and BHI-YH/F-12 medium (BHI/F-12). Bacteria were incubated in the VDC without host cells and OD₆₀₀ was monitored over 6 h (EPEC and L. reuteri) or 23 h (R. gnavus). Mean±s.d., n=4. (D) EPEC A/E lesion formation in T84 cells after 4 h incubation in apical BHI-YH/F-12 medium. Cells and EPEC were stained with F-actin (green) and DAPI (red), respectively. Scale bar: 5 µm. Images are representative of n=6. (E) Optimization of bacterial inocula for co-incubations. EPEC was cultured in the VDC with different ratios of L. reuteri or R. gnavus for 4 h. Culture composition was evaluated by immunofluorescence staining for R. gnavus NanH or L. reuteri CmbA (green) and counterstaining of all bacteria with DAPI (blue). Scale bars: 5 µm. Images are representative of n=6.