Pcs 300 010

Human cell lines used in the in vitro studies included preferred niches of colonization/infection of S. pyogenes from the respiratory track: Detroit 562 (ATCC^® CCL138^™), a cell line derived from the metastatic site of pharynx carcinoma, primary Bronchial/Tracheal Epithelial Cells (BTEC) (ATCC^® PCS300010^™), and A549 (ATCC^® CCL185^™), a cell line derived from a human adenocarcinoma of the alveolar basal epithelial cells. All cell lines were purchased from the American Type Culture Collection (ATCC) (www.atcc.org) and cultured according to the manufacturer′s specifications. Detroit 562 and A549 cultures were maintained in Dulbecco's modified eagle medium (DMEM, ThermoFisher Scientific) supplemented with 10% (v/v) Fetal Bovine serum (ThermoFisher Scientific), and a mixture of 100 U/ml penicillin and 100 μg/ml streptomycin (ThermoFisher Scientific). BTEC were maintained in Airway Epithelial Cell Basal medium (ATCC) supplemented with Bronchial epithelial cell growth kit (ATCC), 33 μmol/l Phenol Red (Sigma) and a mixture of 100 U/ml penicillin and 100 μg/ml streptomycin (ThermoFisher Scientific). For bacterial internalization and adherence assays, human cells were seeded in a 96‐well culture plate (Sigma‐Aldrich Co. LLC, St. Louis, United States of America) at a density of 3 × 10⁴ cells/well and incubated for 24 hours at 37°C, 5% (v/v) CO₂ and 99% (v/v) relative humidity.

Normal human primary bronchial/tracheal epithelial cells (pBECs) were purchased from ATCC (PCS-300-010). The pBECs were transferred to six T25 flasks containing complete Airway Epithelial Cell medium (ATCC) and cells were incubated at 37°C with 5% CO₂ until they reached approximately 80% confluence. Cells were then detached using 0.25% trypsin (Gibco) and transferred to 1 μm transwell permeable supports (Falcon) in a 24-well plate at a density of 3.3 x 10⁴ cells/insert (200 μL per insert). The basolateral side received 700 μL of complete Airway Epithelial Cell medium. Media changes were made every 2d with the apical layer receiving 200 µL and the basolateral layer receiving 700 µL. Once the cells reached 100% confluence, the media was aspirated from the transwell which was then transported to a new 24-well plate and 600 μL of PneumaCultTMALI Maintenance medium (STEMCELL Technologies) was added to the basolateral side. Cells were left to grow for 28d with basolateral media changes occurring every 2d. After approximately 7d, apical washes using 200 μL of DPBS were performed every week to clear the cells of mucus production. After 28d the cells were used for experiments.

Both isolated primary PRECs and commercially acquired HRECs (ATCC, PCS-300-010, Lot-70002486) were subcultured on cell/tissue culture flasks or plates (Greiner Bio-One North America Inc, Monroe, NC, USA), pre-coated with PureCol^® Type I collagen (40 µg/mL/4 mm²; Advanced BioMatrix, Inc., San Diego, CA, USA), at a density of ~20,000 cells/ cm². Both PRECs and HRECs were propagated in ATCC airway epithelial cell basal medium (ATCC^® PCS-300-030™) supplemented with 500 mg/mL HSA, 0.6 mM linoleic acid, 0.6 mg/mL lecithin, 6 mM L-Glutamine, 0.4% Extract P, 1.0 mM epinephrine, 5 mg/mL transferrin, 10 nM 3,3′,5-Triiodo-L-thyronine (T3), 5 mg/mL hydrocortisone, 5 ng/mL rh epidermal growth factor (EGF), 5 mg/mL rh insulin, Pen-Strep and Amp-B (growth media). Cells were dissociated with 0.5X TrypLE^TM express enzyme (Thermo Fisher Scientific) and neutralized using 50% heat-inactivated FBS (EquaFetal™, Atlas Biologicals), mixed in LHC basal medium (Thermo Fisher Scientific). Specifically, primary cells used in this study corresponded to passage 16 for PRECs and 9 for HRECs.

All cell lines were purchased from the American Type Culture Collection (ATCC); A549 (CAT NO. CCL-185), SW480 (CCL-228), lnCAP (CRL-1740), and PC3 (CRL-1435) cell lines were maintained using RPMI-1640 medium (Sigma-Aldrich, R8758). NCI-H460 (HTB-177), HT29 (HTB-38), MCF7 (HTB-22), and MDA-MB-231 (HTB-130) cell lines were kept in high glucose DMEM (Sigma-Aldrich, D5796). Media were supplied with 10% FBS (Biological Industries, 04-007-1A), 100 units/ml of penicillin G and 100 μg/ml of streptomycin (Biological Industries, 03-031-1B). Normal bronchial/tracheal epithelial Cells (PCS-300-010) were grown in serum-free Airway Epithelial Cell Basal Medium (ATCC, PCS 300-030) supplemented with the appropriate cell growth kit (ATCC, PCS-300-040). All cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

LLC-MK2 (ATCC, CCL-7) and A549 cells (ATCC, CCL-185) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 8% fetal calf serum (FCS; Life Technologies), 25mM HEPES (Gibco) and 50 μg/ml Gentamicin (Life Technologies). Primary normal human bronchial/tracheal epithelial (HPBE) cells (ATCC, PCS-300-010) were maintained in airway epithelial cell basal media (ATCC, PCS-300-030) supplemented with bronchial/tracheal epithelial cell growth kit components (ATCC, PCS- 300–040). A549 cells constitutively expressing Rab11-mRFP or Rab8-mRFP were established as described before for HeLa cells [13 (link)]. Briefly, A549 cells were transfected with the mRFP-Rab11a or mRFP-Rab8a cDNA in pCAGGS-HygR vector and hygromycin resistant cells were selected and grown in DMEM containing 8% FCS (DMEM-FCS). SeV (strain Enders), hPIV1 (strain C-35) and hPIV3 (strain C243, ATCC VR-93) were grown in LLC-MK2 cells in DMEM supplemented with 0.15% bovine serum albumin (DMEM-BSA) and acetylated trypsin at 2 μg/ml.