Begm bronchial epithelial cell growth medium bulletkit

Cell lines were obtained from ECACC (SK-GT 4, OE33, FLO-1); ATCC (GO (CP-B)); and Invivogen (THP1-XBlueTM-CD14 (NF-kB/AP-1- Reporter Monocytes)). Cell lines were maintained in culture for no more than 20 passages. Cells were not authenticated in the past year. Cell lines from ECACC were tested for mycoplasma contamination twice per year using PCR as described by Young et al. [19 (link)]. SK-GT 4, OE33, FLO-1 and THP1-XBlue-CD14 were cultured in RPMI 1640 GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA, Catalog no. 61870036) with 10% foetal bovine serum (Labtech, Sorisole, Italy, Catalog no. FCS-SA/500) and penicillin–streptomycin (100 U/mL, 100 µg/mL, Catalog No. 15140148). GO were cultured in BEGM™ Bronchial Epithelial Cell Growth Medium Bullet kit (Lonza, Basel, Switzerland, Catalog no. CC-3170). Where indicated, cells were pre-incubated (1 h) with the TLR2 neutralising antibody (αTLR2 (0.4–40 μg/mL), T2.5, Invivogen) prior to stimulation. Cells were stimulated using Pam3CSK4 (P3C; 0.05 μg/mL, Invivogen, San Diego, CA, USA, tlrl-pms), Pam2CSK4 (P2C; 0.05 μg/mL, Invivogen, tlrl-pm2s-1), recombinant HMGB1 expressed from HEK 293 (stock conc. 50 µg/mL; Sigma-Aldrich, St. Louis, MO, USA; SRP6265), and LPS from Escherichia coli 0111:B4 (1 μg/mL, Sigma-Aldrich, L2630), or with 50% conditioned media (CM), generated as described below.

The human bronchial epithelial cell line BEAS-2B
(CRL-9609) was purchased from ATCC (Manassas, VA). Cells were tested
regularly for mycoplasma contamination. BEAS-2B were cultured using
a BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza, Basel,
Switzerland) at 37 °C in a humidified atmosphere with 5% CO₂. In order to obtain a clonal parental BEAS-2B cell line,
cells were seeded at limited dilution (500 cells per 96-well plate).
Cells were left to recover until they were sufficiently viable to
transfer (4–8 days), after which wells were visually inspected
to identify those containing a single cell. These clones were expanded
and passaged to Primaria (Corning, Corning, NY) 6-well plates and
then to Primaria 10 cm dishes. The clonal parental population was
chosen on the criteria that the cells displayed the same morphology
as BEAS-2B cells and had the same division time. DNA was extracted
(see Section 2.6),
and clonality of the population was confirmed by whole-genome sequencing.
The resulting clonal parental BEAS-2B cell line was cultured in the
same way as the regular BEAS-2B cell line.

WI-38 and HFL1—fibroblast cell lines—were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cells were grown in EMEM medium (WI-38) and HAM’s12 medium (HFL1) with an addition of 10% fetal bovine serum, 2 mM of L-glutamine, 1% of non-essential amino acids, and standard Penicillin Streptomycin solution (Sigma-Aldrich, St. Louis, MO, USA). The epithelial cell line—NHBE—was purchased from Lonza (Lonza Walkersville Inc. Walkersville, MD, USA) and cultured in BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza Walkersville Inc. Walkersville, MD, USA). The experiments (n = 6) were performed after reaching 80–90% confluence (passage three to eight) by the cells. The viability of the cells was assessed using Presto Blue (BD Pharmingen, Franklin Lakes, NJ, USA) and measuring the absorbance at 570 nm.

Fibroblast cell lines—WI‐38 and HFL1—were purchased from Sigma‐Aldrich (St. Louis, MO USA) and grown in EMEM medium (WI‐38) and HAM's12 medium (HFL1) with 10% foetal bovine serum, 2 mM of L‐glutamine, 1% of non‐essential amino acids and standard Penicillin‐Streptomycin solution (Sigma‐Aldrich, St. Louis, MO). The epithelial cell line—NHBE—was purchased from Lonza (Lonza Walkersville Inc. MD, USA) and cultured in BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza Walkersville Inc. Walkersville, MD, USA). All the experiments (n = 6) were performed after reaching 80–90% confluence (passage three to nine) by the cells. The viability of the cells was assessed by adding 10 µl of Presto Blue (BD Pharmingen, Franklin Lakes, NJ, USA), and the absorbance was measured at 570 nm.