Bsa 1

Mouse EpSCs were seeded onto coverslips pre‐coated with type IV collagen, fixed with 70% methanol in acetone and blocked for 1 hour with BSA 1% (Sigma) at ambient temperature. The cells were then incubated with rabbit anti‐mouse MMP9 (GTX100458, GeneTex, 1:400) at 4°C overnight, followed by a one‐hour incubation with phycoerythrin (PE)‐labelled donkey anti‐rabbit second‐class antibody (1:500; Invitrogen) under the same conditions and a five‐min incubation with DAPI (5 μg/mL, Sigma). The stained cells were examined under a laser scanning confocal fluorescence microscope (Leica).

First, 5 × 10⁷ spores of C. difficile 630, C. difficile 630 cotA, C. difficile 630 cotB, C. difficile 630 cotCB were fixed in poly-L-lysine (Sigma-Aldrich, MA, USA)-coated glass cover slides with paraformaldehyde (pH 7.4) for 20 min. Fixed spores were washed three times with PBS, blocked with BSA 1% for 1 h (Sigma-Aldrich, USA) and incubated with primary antibody 1:250 rabbit anti-FLAG-IgG (Rockland 600-401-383). The cover was washed three times with PBS and incubated for 1 h with secondary antibody 1:500 anti-rabbit IgG-Alexa Fluor 488 conjugated (A32731, Invitrogen, MA, USA) and washed three times with PBS. Once the cover dry, it was mounted with the Dako fluorescence mounting medium (Dako Morth America, CA, USA) and sealed with transparent nail polish. Samples were analyzed with a BX53 Olympus fluorescence microscope. The fluorescence intensity was quantified using ImageJ [37 (link)]. Three biological replicates were performed for each C. difficile strain–plasmid combination.

To induce an infection, 20 μL of a bacterial suspension (10³ to 10⁶ CFU/mL) was transferred onto the apical side of the 3D co-culture model. This bacterial concentration is based on our previous infection studies, which we carried out on mice [26 (link),27 (link)], as well as the concentration used in the studies by Charles et al. on tissue-engineered skin to study in vitro biofilm development [17 (link)]. As shown in Figure 1C, 24 h before bacterial inoculation of the co-culture model, the medium was changed to PBS with BSA (1%) (Sigma Aldrich, Taufkirchen, Germany) without antibiotics. The 3D co-cultures were treated with saline as a control or infected with biofilm-forming Staphylococcus. After further cultivation, samples of the surrounding medium and co-cultivated cells were harvested for analysis 3 h, 6 h, 12 h, and up to one day after infection or stored at −80 °C for further investigations. The experimental design of the 3D co-culture model infection is shown schematically in Figure 1.