Penicillin g

Human hepatocellular carcinoma cells (HepG2/C3A, ATCC, ref. number CRL-10741) maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO Life Technologies, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS, Biochrome, Berlin, Germany), 1% of 200 mM L-glutamine (Biochrome), and 1% of antibiotics solution (Penicillin G:10.000 IE/mL/Streptomycin: 10 mg/mL; Biochrome, Berlin, Germany) were used. Cells were routinely incubated under a humidified atmosphere containing 5% CO₂ at 37 °C and were regularly subcultured every 2–3 days.

The preparation of the tracheal organ cultures (TOC) has been described previously [43 (link)]. Briefly, tracheae were dissected from humanely sacrificed 20-day-old specific-pathogen-free (SPF) chicken embryos (Valo BioMedia GmbH, Osterholz-Scharmbeck, Lower Saxony, Germany). They were subsequently cut into equal rings of 0.8 mm and placed separately into 5mL-tubes (Sarstedt AG & Co KG, Nümbrecht, North Rhine-Westphalia, Germany) filled with 1 mL pre-warmed (~37 °C) 199 Hanks’ salts medium (Sigma-Aldrich, Taufkirchen, Bavaria, Germany), supplemented with 1% L-glutamine (200mM, Biochrome, Berlin, Germany) and 1% Penicillin G (10,000 U/mL, Biochrome, Berlin, Germany). The TOCs were placed in an overhead shaker and incubated at 37 °C for four days. After microscopical evaluation, tracheal rings with 100% ciliary activity were randomly assigned to different groups.

The procedure of tracheal organ culture (TOC) preparation was described by Cherry & Taylor-Robinson [36 (link)]. Briefly, tracheas were dissected from 19 days-old specific-pathogen-free (SPF) humanely sacrificed chicken embryos (Valo BioMedia GmbH, Germany). Tracheas were stripped from connective tissue and cut into equal rings (12 / trachea). Each TOC was placed separately in a 5 mL-tube (Sarstedt AG & Co KG, Germany), filled with 1 mL 37 °C pre-warmed Medium 199 Hanks’ salts (Sigma-Aldrich), supplemented with 1% L-glutamine (200 mM, Biochrome, Berlin, Germany) and 1% Penicillin G (10 000 U/mL, Biochrome). The tubes were placed in an overhead shaker and TOCs incubated 4 days at 37 °C. After microscopic evaluation for ciliary beating, the TOCs from each embryo with a 100% activity were split equally in number and allocated to five groups.