Cytometer setup and tracking beads

Whole blood samples were obtained from all children. All samples were obtained between 10:00am and 4:00pm. After collection, blood was allowed to clot by leaving it undisturbed at room temperature and then serum extracted after blood had been processed at 1,000–2,000 x g for 10 minutes in a refrigerated centrifuge. Serum was kept at −80°C until further analyzed. As the samples were labeled with numbers, without any group identification, the investigators were blinded for all procedures.
BDNF serum levels were measured with sandwich-ELISA, using a commercial kit according to the manufacturer's instructions (Milipore, USA). For assessment of oxidative stress, serum levels of malondialdehyde (MDA), a product of lipid peroxidation, were measured by the TBARS (thiobarbituric acid reactive substances) method [51 (link)]. Serum IL6 levels were measured by flow cytometry using the Cytometric Bead Array (CBA) Flex Set Kit (BD Biosciences, San Jose, CA) (Cat. #558276). Acquisition was performed with a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). The instrument has been checked for sensitivity and overall performance with Cytometer Setup and Tracking beads (BD Biosciences) prior to data acquisition. Quantitative results were generated using FCAP Array v1.0.1 software (Soft Flow Inc., Pecs, Hungary).

Patient tissue was dissociated by using the human Tumor Dissociation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany, 130-095-929) and the GentleMACS system (Miltenyi Biotech). Sample acquisition was done on a LSRII/Fortessa flow cytometer (BD, San Jose, CA, USA) expressed as mean fluorescence intensity (MFI). Both antibodies and secondary reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-colour compensation to create multi-colour compensation matrices. For gating, fluorescence minus one (FMO) controls were used. Cytometer Setup and Tracking beads (BD) were used for daily control of instrument calibration. Staining of patient-derived single-cell suspensions contained CD45 AF700 (Biolegend, San Diegeo, CA, USA, 368513) to exclude immune cells and CD326 PE-CF594 (BD Biosciences, San Jose, CA, USA, 565399) to gate CD326⁺ healthy and tumour epithelial cells, respectively. LCN-2R (Thermo Fisher, PA5-20543) was stained intracellularly in combination with an AF546-labelled secondary antibody (Life technologies, A-11035), after fixation and premeabilisation with Cytofix/Cytoperm (BD, 554714).

For FACS-sorting of fibroblasts, tissue single suspensions were generated using the gentleMACS dissociator and the mouse Tumor dissociation kit (both from Miltenyi Biotec, Bergisch Gladbach, Germany). Single cell suspensions were stained with fluorochrome-conjugated antibodies and sorted using a FACS Aria III cell sorter (BD Biosciences, Heidelberg, Germany). Data were analyzed using FlowJo software Vx (BD Biosciences, Heidelberg, Germany). Antibodies and secondary reagents were titrated to determine optimal concentrations. CompBeads (BD Biosciences) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one (FMO) controls were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking beads (BD Biosciences). The following antibodies were used: anti CD49f-PE-CF594, anti-CD140b-PE, anti-CD140a-APC, anti-CD326-BV711 (BD Biosciences), anti-CD31-PE-Cy7 (eBioscience, Frankfurt, Germany), and anti-CD45-VioBlue (Miltenyi Biotec). 7-AAD was used for dead cell exclusion.

Staining antibodies, stimulation reagents, media, and other reagents were purchased from BD Bioscience (Franklin Lakes, NJ, USA), BioLegend (San Diego, CA, USA), eBioscience/Thermo Fisher (Carlsbad, CA, USA), Illumina (San Diego, CA, USA), Invitrogen/Thermo Fisher (Carlsbad, CA, USA), Life Technologies/Thermo Fisher (Carlsbad, CA, USA), Miltenyi Biotec (Bergisch Gladbach, Germany), Jackson ImmunoResearch (West Grove, FL, USA), PeproTech (Rocky Hill, NJ, USA), Promega Corporation (Madison, WI, USA), Selleck Chemicals (Houston, TX, USA), Sigma-Aldrich (St. Louis, MO, USA), and UCB Pharma (Slough, UK) and listed in Tables S2, S3. Quality control of flow cytometry stainings was performed using SPHERO Rainbow Calibration Particles (BD Bioscience, Franklin Lakes, NJ, USA) and Cytometer Setup and Tracking beads (BD Biosciences, Franklin Lakes, NJ, USA) for stable MFIs over time (57 (link)).

As NTHi is difficult to distinguish from H. haemolyticus by standard microbiological culture, we used the fluorescent dye Cell Trace Violet (CTV, excitation = 390 nm, emission = 445 nm; Invitrogen) to label NTHi86-028NP for experiments involving bacterial co-culture. Overnight growth of bacteria was harvested from chocolate agar plates and resuspended in cell culture media to an OD_600nm 0.2 (which is equivalent to ~10⁸ CFU/mL). Bacterial suspensions were incubated with 20 μM CTV for 5 min before pelleting at maximum speed in a benchtop centrifuge for 7 min. Harvested bacteria were resuspended in 10 mL of pre-warmed epithelial cell culture media, protected from light and incubated at 37°C for 10 min before use. The CTV stain did not affect the ability of NTHi86-028NP to attach to or invade A549 cells (data not shown). Flow cytometry was conducted on a Flow and cell cytometer Canto II device (BD Biosciences) that was calibrated using Cytometer Setup and Tracking beads (BD Biosciences). Forward scatter and side scatter were set to capture bacterial populations. Flow cytometry data was acquired using FACSDiva software and then analyzed using Flow Jo v7 software (FloJo LLC, Oregon, USA).