Humidified incubator

Specimens obtained during surgery were directly placed on ice in Dulbecco modified Eagle medium (PAA, Pasching, Austria) supplemented with penicillin and streptomycin. For tissue culture, specimens were sliced (200μm thickness) using McIlwain tissue chopper (Ted Pella, Inc., Redding, CA) before being transferred to culture plate inserts comprising a 0.4 μm nitrocellulose membrane (Millipore/Greiner, Billerica, MA, USA). Slice-containing membranes were placed at the interface of air and B-ALI differentiation medium (Lonza, Basel, Switzerland) within respective cell culture well-plates (TPP Techno Plastic Products, Trasadingen, Austria) and DMEM High Glucose (Biochrom, Berlin, Germany) followed by culture in a humidified incubator (Binder, Tuttlingen, Germany) at 37°C and 5% CO₂. Nasal slices were cultured for up to 4 weeks and fed every two days by replacing the medium with fresh B-ALI differentiation medium.
Human nasal slices cultivated for 7 days were treated with LPS (100 ng/ml, rough strains from Salmonella enterica Re 595, cat. no. L9764, Sigma-Adrich St. Louis, MO, USA) or LPS and 1,8-Cineol (10⁻⁵ M, Klosterfrau Healthcare Group, Cassella-med GmbH & Co. KG, Cologne, Germany as described in [24 (link)] for 60 minutes followed by respective stainings or real time PCR.

Human embryonic kidney (HEK293; female; RRID: CVCL_0045) and NIH3T3 (male; RRID: CRL-1658) cells were obtained from DSMZ (Braunschweig, Germany) and ATCC (American Type Culture Collection), respectively. HEK293 and NIH3T3 cells were cultivated in DMEM culture medium (DMEM supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin, 4 mM L-glutamine) in a humidified incubator (BINDER) at 37°C and 5% CO₂. To maintain a confluency of 70–90%, cells were split at a 1:7 to 1:10 ratio every 2 to 3 days. For this, the cells were washed with PBS (without CaCl₂ and MgCl₂), followed by addition of trypsin-EDTA solution (Sigma) and incubation at 37°C until cells detached. Afterward, cells were resuspended using culture medium and split in the desired ratio. For storage, cells were harvested at 300 g for 5 min, resuspended in freezing medium (90% FBS, 10% DMSO) and gradually frozen to −80°C. Cells were routinely checked for mycoplasma contamination by PCR.⁵³