Tepc 15

Bacterial strains grown in TS broth at 37°C to an OD_620nm of 1.0 were washed and diluted in 1% PBS-BSA to a final concentration of 10⁶ CFU in 100 μL. Bacteria were stained with the mAb to phosphorylcholine, TEPC-15 (Sigma-Aldrich, 1:5000 dilution) followed by a FITC-labeled antibody to murine IgA. Reactions were carried out at 4° C for 30 mins without spin or wash steps in between. After staining, samples were fixed with 4% paraformaldehyde (PFA), washed once with PBS, and resuspended in PBS. The surface shielding assay was also performed using ex vivo samples of bacteria collected from colonized mice. T6A strains P376 or P385 were administered IN to 10-day-old pups at a dose of 10⁶ CFU in 3 μL. Two hours post-inoculation, pups were euthanized, and URT lavages collected. Lavages were pooled from at least 6 pups, pelleted and resuspended in 100 μL and treated as described above. Samples were analyzed by flow cytometry using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

For detection of glycan epitopes, protein extracts of adult worms, both female and male were analysed. The worms were cut into pieces of ~2 mm and homogenised using a Dounce homogeniser in lysis buffer (20 mM MES buffer pH 7, 1% Triton X-100, 0.01% protease inhibitor cocktail from Roche). The homogenate was then sonicated for 3 s, centrifuged at 10,000×g for 10 min and the supernatant denatured at 95 °C in 4× sample buffer (60 mM Tris–Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) prior to SDS-PAGE separation (8% acrylamide, 120 V) for subsequent analysis by either Coomassie staining or blotting on nitrocellulose (GE Healthcare). After overnight blocking of the membrane at 4 °C with 0.5% bovine serum albumin in PBST, lectin or antibody detection was performed using either 10 μg/ml of biotinylated lectins (CGL3 or CCL2) or 1:5000 dilution of the murine IgA TEPC 15 (Sigma, M1421). Subsequently the membrane was incubated with, in the lectin case, 10 μg/ml of Streptavidin coupled with HRP (Vector Laboratories, SA-5004) and in the TEPC 15 case a 1:3000 dilution of HRP coupled anti-mouse IgA (Bethyl, A90-103P). After extensive washing (PBST), horseradish peroxidase activity was detected using ECL Direct™ detection reagent (VWR, RPN2105) and exposure to photographic film.

Three to four colonies of NTHi grown on chocolate agar for 16 h were inoculated into 20 ml sBHI, grown for 11 h, diluted into 40 ml sBHI to OD₆₀₀ = 0.05, grown to OD₆₀₀ = 0.6 in the absence or presence of thymidine, serially diluted, plated on sBHI agar for c.f.u. determination, and used to generate stocks stored at −80°C in sBHI with 20% glycerol as single use aliquots for further experiments. For PCho determination, ~1 × 10⁷ c.f.u. were incubated for 1 h at 37°C with TEPC-15, a mouse monoclonal antibody specific for PCho (Sigma-Aldrich) diluted 1:25 in PBS-0.05% Tween 20. Samples were washed twice with PBS-0.05% Tween 20, and incubated with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse (Serotec) diluted 1:300 in PBS-0.05% Tween 20 for 30 min at 4°C under dark conditions. Bacteria were washed with PBS-0.05% Tween 20, fixed in 3% paraformaldehyde (PFA) for 2–3 min at room temperature, and analyzed on a FACSCalibur flow cytometer (BD Biosciences) using forward and side scatter parameters to gate on at least 25,000 bacteria. Results are expressed as a relative percent fluorescence index (RFI), to measure both the proportion of fluorescent bacteria positive for PCho and the intensity of fluorescence (Ramos-Sevillano et al., 2015 (link)). Assays were performed in quadruplicate in at least three independent occassions (n ≥ 12).