Porcine type 2 mucus

Strains were grown overnight in the presence of ³H-labeled thymidine (Perkin Elmer). Cultures were then incubated in a Maxisorp 96-well plate coated with either porcine type II mucus (Sigma-Aldrich) or human mucus, followed by extensive washing with PBS (Oxoid) and lysis of the remaining adherent cells. Sample radioactivity was measured using a Wallac 1480 Wizard 3 automatic gamma counter (Perkin Elmer). Full experimental details are described in Vesterlund et al. (2005) (link). Each assay was performed in three biological repeats, with each a minimum of three technical replicates per strain.

Mucus adherence of the strains was measured as described previously (Vesterlund et al. 2005 (link); Rasinkangas et al. 2014 (link)). In short, strains were grown overnight in the presence of ³H-labeled thymidine (Perkin Elmer). These cultures were then incubated in a Maxisorp microtiter plate coated with porcine type II mucus (Sigma). Wells were washed with PBS (Oxoid) and the remaining adherent cells were lysed. Radioactivity of the samples was measured with a Wallac 1480 Wizard 3 automatic gamma counter (Perkin Elmer). Three repeats of the mucus binding assay were performed, and in each assay, 3–6 technical replicates were prepared for each strain. Data analysis was performed using GraphPad Prism® 8. Significant differences between two groups were calculated using unpaired t tests, and the significance level was set at p < 0.0001.