Chloroform

Extracellular glycolipids were extracted as previously described [28 (link)]. Glycolipids (MELs and CLs) were separated by thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany) first with a solvent system consisting of chloroform–methanol–water (65:25:4, v/v/v) (Roth, Karlsruhe, Germany) for 5 min followed by a second solvent system consisting of chloroform–methanol (9:1, v/v; 2 × 18 min) [35 (link)]. The plates were dried, and sugar containing compounds were visualized by application of a mixture of ethanol:sulfuric acid:p-anisaldehyde (18:1:1, v/v) (Roth, Karlsruhe, Germany; Merck, Darmstadt, Germany; Sigma Aldrich, Steinheim, Germany) followed by heating at 150 °C for 2 min [36 (link)].

To assess mucus type 2 layer thickness, three wells of host cells per tested donor (and per blank) were fixated (after host-microbe coincubations) in Carnoy’s reagent, consisting of 60% ethanol (≥ 99.9%, Sigma-Aldrich, Merelbeke, Belgium), 30% chloroform (≥ 99%, Carl Roth, Karlsruhe, Germany) and 10% glacial acetic acid (≥ 99.7%, Sigma-Aldrich, Merelbeke, Belgium) for 30 min at 4 °C. After three washing steps, 3% (w:v) BSA in PBS was used as blocking solution overnight at room temperature. Blocking solution was removed, 1 µg/mL MUC2 primary polyclonal antibody (PA5-79,702, Invitrogen, Carlsbad, USA) was added and incubated overnight at 4 °C. After three washing steps, 5 µg/mL Alexa Fluor 488 conjugated secondary antibody, (A-11034, Invitrogen, Carlsbad, USA) was incubated for one h at room temperature. Following three washing steps, imaging was performed with a Nikon A1R confocal microscope equipped with a Plan Fluor 40x/0.6 objective (Nikon Instruments Amsterdam, the Netherlands). In each well, z-stacks (slices every 1.25 µm) of mucus layers were recorded at four different positions. To determine the mucus layer thickness, FIJI software (https://imagej.net/Fiji) was used with an in-house developed script (macro) as described by Beterams et al. ^{28 (link)}.