Phosphate buffered saline (pbs)

Cells grown on coverslips were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS (Sigma-Aldrich) for 20 min, permeabilized in 0.3% Triton X-100 (Sigma-Aldrich) for 15 min, washed three times with PBS (Sigma-Aldrich) and blocked with PBS (Sigma-Aldrich) containing 5% normal goat serum (Sigma-Aldrich) and 0.3% Triton X-100 (blocking buffer) for 1 h at RT. Cells were incubated overnight at 4 °C with primary antibody NG2 (Santa Cruz Biotechnology) diluted 1:50 in blocking buffer, washed three times with PBS (Sigma-Aldrich) and then incubated with goat anti-rabbit IgG AF568-conjugated secondary antibody (Thermo Fisher Scientific) diluted 1:1000 in blocking buffer for 1 h at RT. Finally, the nuclei were stained with DAPI (Roche) for 10 min. Images were acquired using a Leica TCS SP8 X confocal microscope with HCX PL APO 63x/1.4 objective. Confocal z-stacks were acquired with sections of 0.3 µm.

A commercially available extracellular-type trehalose-containing Kyoto (ETK) solution (Otsuka Pharmaceutical Factory, Naruto, Japan) supplemented with 5 μM alvelestat was used as preservation solution. Dimethyl sulfoxide (DMSO; Sigma-Aldrich Japan) plus phosphate-buffered saline (PBS; Sigma-Aldrich Japan) was used to dissolve alvelestat (alelestat(+)), and DMSO plus PBS without alvelestat (alvelestat(−)) was used as the vehicle control.

DNA extraction was done using the Saponin-Chelex extraction method. Saponin (0.5%) in phosphate buffered saline (PBS) was prepared by dissolving 1 g of saponin and one tablet of PBS (Sigma-Aldrich, USA) in 200 ml of sterile distilled water, shaken vigorously and allowed to stand till the saponin and PBS dissolved completely. Each of the dried blood spot was cut out from the DBS papers with sterilized scissors with serrated edges and each of the discs were transferred to pre-labelled Eppendorf tubes. 1 ml of the saponin-PBS solution was added to the specimen in the Eppendorf tubes and allowed to stand for 24 h at 4 °C for complete haemolysis.
The specimens were washed three times in PBS with intermittent centrifugation at 13,000 rpm for 5 min after each wash. Subsequently, 1 ml of PBS was added to the sediment, and then it was vortexed for 1 min and incubated for 30 min at 4 °C. It was centrifuged again for 5 min at 13,000 rpm and the supernatant decanted.
Fifty microlitres of freshly prepared Chelex solution was added to the tubes followed by 150 μl of distilled water to obtain 5% of the Chelex solution. It was then vortexed and incubated in a block heater set at 99 °C for 20 min, centrifuged at 13,000 rpm for 5 min and the supernatants decanted into newly labelled Eppendorf tubes. The supernatants contain the DNA.

Resistance of bacterial strains to acid and bile salts was assessed using the method adapted from Kechaou et al. (2013 (link)). Bacteria from overnight cultures were washed twice with PBS, pH = 7.5 (Lonza, Verviers, Belgium), and diluted to OD_600 nm = 0.5 in 1 ml of PBS, pH = 7.5 (control); PBS, pH = 3 (acid stress); and PBS, pH = 7.5, containing 3 g l⁻¹ of two bile salts: sodium cholate and sodium deoxycholate (Sigma-Aldrich, St. Louis, MO, USA) (bile salt stress). After incubation for 1 h at 37 °C, bacterial cells were washed with PBS and suspended in M17-glucose (0.5% w/v) medium. For each strain and each condition, four 2-fold serial dilutions were prepared in duplicate on a sterile 384-well microtiter plate (Greiner Bio-One GmbH, Frickenhausen, Germany) starting from OD_600 nm = 0.025. Growth of bacteria at 37 °C was monitored every 15 min for 28 h by measuring the OD_600 nm with the Infinite M200 Pro plate reader (Tecan, Maennedorf, Switzerland). The resistance of bacterial strains to digestive stress was determined by measuring the growth delay (i.e. delay in time needed to reach mid-exponential phase) between stressed and non-stressed cultures. For each strain and each condition, four growth delays corresponding to the four dilutions on the microtiter plate were averaged.

Murine monocytes were isolated from the spleen. Spleen tissue of sacrificed mice was squeezed through a 40 µm mesh filter and the filter was subsequently washed with PBS (Sigma Aldrich Co., St. Louis, MO, USA). The obtained cell suspension was processed through differential gradient centrifugation in Ficoll gradient (922 × g, 18 min). The resulting white intermediate layer was resuspended in PBS (Sigma Aldrich Co., St. Louis, MO, USA) and centrifuged (535 × g, 10 min) again. To lyse erythrocytes, the obtained cell pellet was resuspended in distilled water for 30 s. The addition of PBS (Sigma Aldrich Co., St. Louis, MO, USA) and centrifugation (535 × g, 10 min) was followed by adhesion depletion on a plastic surface. Monocytes were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37 °C and 5% CO₂ in a humidified atmosphere. Non-adherent cells were removed by gentle washing with PBS on the following day, and the remaining adherent cells were harvested.

Total BALBc or Il1rl1^−/− PLEC were labelled with 5 μg ml⁻¹ CFSE (Invitrogen) or 2 μM CellTrace Violet (Molecular probes), respectively, before injection of 200,000 labelled cells at a 1:1 ratio into the pleural cavity in 100 μl PBS. Fifty μg of Alt was delivered i.n. 18 h later. To block IL-5 within the pleural cavity, 30 μg of either purified functional grade anti-human/mouse IL-5 (eBioscience, clone TRFK5), 30 μg of Rat IgG1 (eBioscience, clone eBRG1) in 100 μl PBS (Sigma), or PBS (Sigma) alone was injected i.pl., immediately following injection 50 μl PBS or 50 μg of Alternaria extract (Greer) in 50 μl PBS (Sigma) was delivered i.n.