Cell scraper

Macrophages were mechanically harvested after 7 to 12 days of differentiation/treatment using a cell scraper (Greiner Bio-one, Kremsmünster, AT), spun at 1000 rpm/10 min, and analyzed for cell integrity and viability by microscopy using Trypan blue exclusion or a Live/Dead cell viability dye (Ghost Dye^TM Violet 510, Tonbo Biosciences, San Diego, CA, USA). For immunophenotyping, viable cells were stained using specific fluorophore-conjugated anti-human monoclonal antibodies (Table 1) at concentrations recommended by the manufacturer. Cells were incubated with Abs for 30 min at +4 °C in the dark, washed using PBS/0.1% BSA (FACS Wash) buffer, and fixed in 4% Paraformaldehyde (Biolegend, San Diego, CA, USA) before analyzing at BD FACS Verse^TM flow-cytometer (BD Biosciences, San Diego, CA, USA). Live/dead dye was routinely applied to exclude dead cells before analyzing cells of interest. Data were analyzed using FACS Suite or Diva software v 9.0 (BD Biosciences, San Diego, CA, USA).

Droplets (10 μL) of same bacterial suspensions were dried in the same way as for instantaneous rehydration. After the drying, cells were rehydrated by addition of 990 μL of concentrated PBS (noted cPBS, a_W = 0.950, checked using an a_W-meter) and homogenized by pipetting and using a cell scraper (Greiner, Les Ulis, France), corresponding to the first rehydration step. The use of cPBS (a_W = 0.950) resulted in the partial rehydration of bacterial cells up to an a_W of 0.950 (compared to PBS with a_W = 0.995) imposing a supplementary step before total rehydration of the bacterial cells at an a_W of 0.995. After 30 s, for the second step, dilution was performed in PBS (a_W = 0.995) and counted by plating on TSA (a_W = 0.995) incubated for 24 h at 37°C (+/‒ 0.1°C) (Fig 1(B)).

Mice of both strains were killed by cervical dislocation following anesthesia with pentobarbital. The entire length of the esophagus was dissected and placed in cold (4 °C) PBS lacking Ca²⁺ and Mg²⁺. The muscle layer was removed using forceps and the remaining inner tissue containing the keratinous layer was incubated in 1 ml 0.25% trypsin solution (Invitrogen) at 4 °C for 8 h. The gelatinized submucosal layer and lamina propria mucosae were gently peeled away and the keratinocytes were harvested with a cell scraper (Greiner Bio-one). The obtained cells were filtered with a cell strainer (BD Falcon) and resuspended to a density of 1 × 10⁶ cells/ml and seeded in 2-well culture inserts placed on glass bottom dishes (Matsunami, Tokyo, Japan). The cells were incubated in MCDB 153 medium^{13 (link)} containing 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 14.1 μg/ml phosphorylethanolamine, 10 ng/ml epidermal growth factor (all from Sigma), 10 μg/ml transferrin (Funakoshi, Japan), 40 μg/ml bovine pituitary gland extract (Kyokuto, Japan), 25 μg/ml gentamicin, 50 U/ml penicillin, 50 μg/ml streptomycin and 10% fetal bovine serum (FBS). After incubating cells at 37 °C in 5% CO₂ for 24 h, the medium was changed to a medium lacking FBS. Medium with freshly added agonist or antagonist was changed every day as dictated by the experimental design.

After 7, 14 and 21 days in differentiation medium cells were trypsinized (Trypsin-EDTA, Sigma-Aldrich) and treated with a cell scraper (Greiner Bio-One). Cell count and viability were determined by haemocytometer using trypan blue exclusion and proliferation rate was calculated for each drug concentration and control flask, respectively.

For each experiment, a droplet (10 μL) of bacterial suspensions was spread in a thin layer onto a glass Petri dish (with 3 cm diameter) which was placed at 58%, 44%, 25% and 11% RH during 90 min. After the desiccation, cells were rehydrated by instantaneous (1 s) addition of 990 μL of PBS (a_W = 0.995, checked using an a_W-meter) and homogenization by pipetting and using a cell scraper (Greiner, Les Ulis, France). Then, cells were diluted in PBS (a_W = 0.995) and counted by plating on TSA (a_W = 0.995) incubated for 24 h at 37°C (+/‒ 0.1°C) (Fig 1(A)).

Feces were collected in Fast Prep lysing Matrix A tubes (MP Biomedicals), resuspended in 1 ml of PBS per 100 mg fecal material, and incubated at 4°C for 20 min. Colon mucosal samples were isolated by removing feces and loose material associated with the colon tissue and scraping along the length of a colon with a cell scraper (Greiner Bio One) into fresh cold PBS. Bacterial suspensions were resuspended in a final volume of 2 ml PBS and incubated at 4°C for 20 min. Samples were homogenized in a FastPrep-24 Tissue homogenizer (MP Biomedicals) for 30 s. After homogenization, samples were centrifuged at 50 × g for 15 min at 4°C to remove debris and the bacteria-containing supernatant transferred through 70 µm filters into a new tube. Bacteria were washed in FACS buffer (PBS, 2% FCS, and 5 mM EDTA) and pelleted at 8,000 × g for 5 min. For flow cytometry, bacterial pellets were resuspended in 100 µl FACs buffer containing SYTO 9 green fluorescent nucleic stain (10 µM; Life Technologies), incubated at 4°C for 15 min, and subsequently stained with 1 µg/ml of an anti-mouse IgA-PE antibody (eBioscience) for 30 min at 4°C. Samples were thoroughly washed before acquisition on the flow cytometer (BD Fortessa).

The OD₅₉₅ of the appropriate ON cultures was measured and corrected to an OD₅₉₅ of 3.2. The corrected cultures were diluted by transferring them into 10 ml TSB 1/20 to obtain an initial cell density of ±12 × 10⁷ ml⁻¹. This suspension was poured into small petridishes (Ø 60 mm) and incubated under static conditions at 25 °C. During incubation, the cells attached to and formed a biofilm layer on the bottom of the plates. After 48 h of incubation the liquid medium above the biofilms was removed and 1 ml of phosphate-buffered saline (PBS; 1.24 g l⁻¹ K₂HPO₄, 0.39 g l⁻¹ KH₂PO₄, 8.8 g l⁻¹ NaCl) was added after which the biofilm layers were scraped off with a cell scraper (Greiner). For the phenotypic switch experiment (Supplementary Fig. 1) parallel biofilm plates were set up and the biofilms formed on the bottom and the side of the petridishes were scraped off every 2 h during 24 h. The biofilm layers were passed five times through a syringe (0.5 × 1.6 mm) and vortexed to disrupt cell clumps and obtain single cells. Biofilm and/or planktonic cells were plated out to determine the number of colony forming units (CFU). Three parallel biological repeats (n) were performed, each starting from separate ON cultures.