Reagentpack subculture kit

HRMECs were detached using the ReagentPack subculture kit from Lonza. Firstly, the medium was removed, and the cells were washed with HEPES pre-warmed to 37 °C. Thereafter, trypsin, pre-warmed to 37 °C, was added to the cells for a period of 1 min, detaching the cells and finally, trypsin-neutralizing solution was added. The cells were allowed to recover for a period of 1 h at room temperature in medium supplemented with 10% (v/v) FCS. To exclude dead cells from the analysis, cells were incubated with Zombie Aqua viability dye (BioLegend, San Diego, CA, USA) in PBS for 15 min at room temperature. In addition, the cells were simultaneously treated with human FcR blocking reagent (130-059-901; Miltenyi Biotech, Westphalia, Germany). Afterward, the cells were stained with the Fluorescein isothiocyanate-labeled mouse anti-human CD206 (catalog number 551135 clone 19.2; BD Biosciences, San Jose, CA, USA) in fluorescence-activated cell sorting (FACS) buffer (PBS, 2% (v/v) FCS, 2 mM ethylenediamine tetraacetic acid) for 30 min on ice. The cells were subsequently washed twice in FACS buffer and fixed with 0.4% (v/v) formaldehyde in FACS buffer. Data acquisition was carried out using an LSRFortessa X-20 cell analyzer (BD Biosciences), and data analysis was conducted with FlowJo software, version 10.8.1. (Tree Star, Ashland, OR, USA).

Normal human bronchial epithelial cells (NHBE) of non-smoking subjects were obtained from Lonza (Lonza, Slough, UK) and grown in bronchial epithelial cells growth medium (BEGM) supplemented with growth supplements (SingleQouts kit, Lonza) as recommended by the manufacturer. Cells were passaged at passages 2–8 using the ReagentPack subculture kit (Lonza) following suppliers instructions. Cells were cultured until 80% confluent at 37°C and 5% CO_2. Prior to experiments, monolayers of cells (70–80% confluence) were incubated in basal medium (supplement free) overnight. Cells were treated with BET inhibitors (JQ1 and PFI-1) prior to stimulation with IL-1β (1 ng/ml) in the presence or absence of H₂O₂ (100 µM). All experiments were performed at least four times.