Freshly generated cigarette smoke extract conditioned media was prepared as previously described [49 (link), 50 (link)]. Cigarette smoke from a University of Kentucky 3R4F cigarette was bubbled through 4 ml of serum-free medium, followed by filtration through a 0.22 μm filter, and adjustment with fresh media to an absorbance of 0.15 at 320 nm, to generate a 100% cigarette smoke extract solution. A 4% solution of cigarette smoke extract was made from the 100% solution for use in our experiments. Allopurinol, MK-571, dexamethasone, and cycloheximide were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Amplex Red uric acid analysis kits and epidermal growth factor (EGF) were purchased from ThermoFisher (Burlington, Ontario, Canada). Ferric ammonium sulfate and H2O2 were purchased from SigmaAldrich (Oakville, Ontario, Canada). Recombinant TNF-α and IFN-γ were purchased from Peprotech (Dollard des Ormeaux, Quebec, Canada). Bronchial epithelial cell growth media was purchased from Lonza (Mississauga, Ontario, Canada). Keratinocyte serum free growth media was purchased from Invitrogen (Burlington, Ontario, Canada).
Bronchial epithelial cell growth media
Manufactured by Lonza
Sourced in Canada, Switzerland, United States
Bronchial Epithelial Cell Growth Media is a specialized culture medium designed to support the growth and maintenance of human bronchial epithelial cells in vitro. It provides the necessary nutrients and growth factors to facilitate the expansion and proliferation of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Cayman Chemical (1 mentions)
Ferric ammonium sulfate, by Merck Group (1 mentions)
Mk571, by Cayman Chemical (1 mentions)
Dexamethasone (dex), by Cayman Chemical (1 mentions)
Allopurinol, by Cayman Chemical (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Recombinant tnf α, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Begm bullet kit, by Lonza (1 mentions)
Thle 2, by American Type Culture Collection (1 mentions)
Sk mes 1, by Thermo Fisher Scientific (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Mirna nc mimic mir nc, by RiboBio (1 mentions)
Mir 125b 5p mimic, by RiboBio (1 mentions)
Mir 125b 5p inhibitor, by RiboBio (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
7 protocols using bronchial epithelial cell growth media
1
Cigarette Smoke Extract Conditioning
2
Isolation and Culture of Primary Human Airway Epithelial Cells
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Primary human airway epithelial cells were isolated from bronchial brushings from healthy subjects and those with physician diagnosed asthma and COPD based on ATS/ERS guidelines as previously described [59 (link), 60 (link)]. Briefly, bronchial brushes were collected into 5ml of warmed bronchial epithelial cell growth media in a 15 ml conical tube. The brushes and media were vortexed for 30 seconds to remove cells from the brush. The brush was then removed and washed via pipette with the media in the conical tube to remove as many cells as possible. Cells were then centrifuged (1200 rpm for 7 minutes) for isolation, re-suspended in fresh bronchial epithelial cell growth media, and plated into 24 well culture dishes with 500μL of media. Subject characteristics are outlined in Table 1 . Primary cells were grown under submerged monolayer conditions in bronchial epithelial cell growth media (Lonza—Switzerland).
To complement primary human cell experiments, we used a previously validated minimally immortalized human airway epithelial cell line (HBEC-6KT) grown under submerged monolayer conditions in keratinocyte serum free media with 50ng/ml of bovine pituitary extract and 0.6ng/ml of epidermal growth factor [26 (link), 61 (link)–64 (link)]. All experiments with primary human airway epithelial cells and HBEC-6KT were performed at 90–95% confluency.
To complement primary human cell experiments, we used a previously validated minimally immortalized human airway epithelial cell line (HBEC-6KT) grown under submerged monolayer conditions in keratinocyte serum free media with 50ng/ml of bovine pituitary extract and 0.6ng/ml of epidermal growth factor [26 (link), 61 (link)–64 (link)]. All experiments with primary human airway epithelial cells and HBEC-6KT were performed at 90–95% confluency.
3
Impact of Aspergillus fumigatus on Bronchial Epithelial Cells
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Human primary bronchial epithelial cells (BECs) were purchased from Promocell (Heidelberg, Germany) and Lonza (Basel, Switzerland). Cells were cultured in Bronchial Epithelial Cell Growth Media supplemented with BEGM BulletKit (Lonza) in 75‐cm2 flasks until they reached 80% confluence. For experiments, BECs were used between passages 2 and 3 and seeded at 15 × 103/cm2. Monolayers were exposed to 1 × 105 spores/mL for 12 and 24 hours or 1 μg/mL A fumigatus culture filtrate for 24 hours. At the end of the study, culture supernatants were collected and levels of TGF‐β1, ET‐1, periostin and TGF‐β2 determined using DuoSet® ELISA kits performed according to manufacturer's instructions (R&D Systems, Abingdon, UK). For cultures involving germinating spores, cell layers were collected for analysis of gene expression, whilst supernatants were filtered through a 0.22 μm filter for ELISA.
In some experiments, in order to assess the growth of A fumigatus in the presence of epithelial cells, cultures were stained for calcofluor‐white (Sigma‐Aldrich, Poole, UK) and time‐lapse imaging performed using the Nikon Eclipse TE2000E microscope at X20 using an ORCA‐ER CCD camera (Hamamatsu, Welwyn Garden City, UK).
In some experiments, in order to assess the growth of A fumigatus in the presence of epithelial cells, cultures were stained for calcofluor‐white (Sigma‐Aldrich, Poole, UK) and time‐lapse imaging performed using the Nikon Eclipse TE2000E microscope at X20 using an ORCA‐ER CCD camera (Hamamatsu, Welwyn Garden City, UK).
4
Isolation and Characterization of Nasal Epithelial Cells
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Tissue specimens were obtained during surgery, from nasal polyps and ethmoid tissue of patients with CRSwNP and CRSsNP, respectively. The samples were immediately placed in BEGM medium (Bronchial Epithelial Cell Growth Media, Clonetics-Lonza) with no serum, supplemented with penicillin 100 U/ml, streptomycin 100 µg/ml, and glutamine. Afterwards the samples were washed twice in NaCl solution and cut into small pieces (~1 mm2). Diced specimens were then plated (density, 9 pieces/6-well tissue culture dish) in BEGM medium and incubated in a humidified 5% CO2 atmosphere at 37°C, until a monolayer of epithelial-like cells was observed to be confluent, usually after 13 ± 2 days. Then the explanted tissues were removed, and cells were trypsinized and replated into 6-well tissue culture dish at a final volume of 1.5 ml of fresh BEGM medium. The medium was changed every 3 days for 2-3 weeks until 90% confluence was obtained, usually after 17 ± 3 days, when the cells were stimulated overnight. To assess purity of the culture, cells were trypsinized and washed and one aliquot was analyzed for EpCAM (epithelial cells marker) expression with flow cytometry technique and another aliquot was examined in the Bürker chamber for counting the number of cells.
5
Characterization of HCC Cell Lines
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SNU-387 HCC cells were a generous gift from Dr. Terence Gade (Department of Radiology, University of Pennsylvania). HepG2 HCC cells were obtained from ATCC (Manassas, VA). STR profiling performed at our institution indicated that both cell lines matched reference standards per ATCC guidelines. Differentiation status of both cell lines was based off transcriptomic classifications by Caruso et al., with HepG2 cells characterized by well-differentiated, with hepatoblast-like features and SNU-387 as less-differentiated, invasive, with mesenchymal-like features29 (link). SNU-387 and HepG2 cells were maintained in Roswell Park Memorial Institute (RPMI) (Gibco Life Technologies, Carlsbad, CA) and Eagle’s Minimum Essential (EMEM) media (Corning Life Sciences, Tewksbury, MA) respectively, both supplemented with 10% fetal bovine serum (Gibco) and 0.5% penicillin/streptomycin. The normal human liver epithelial cell line THLE-2 was obtained from ATCC (Manassas, VA) and maintained in Bronchial Epithelial Cell Growth media (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (Gibco) and 0.5% penicillin/streptomycin. All cell lines were maintained in a jacketed humidified CO2 (5%) incubator at 37 °C and passaged when confluent. Core facility testing at our institution for contaminants such as mycoplasma was negative.
6
Transfection and Cell Culturing of BEAS-2B and NSCLC Lines
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The human nontumorigenic bronchial epithelial cell line BEAS-2B (ATCC, Manassas, VA, USA) was maintained in bronchial epithelial cell growth media (Lonza, Walkersville, MD, USA). The NSCLC cell lines A549, SK-MES-1, and H460 were cultured in RPMI-1640, F-12K (Gibco, Thermo Fisher Scientific, Inc.), and Minimum Essential Medium (Gibco) media, respectively. All the NSCLC cell lines were purchased from ATCC. Additionally, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixed reagent (Gibco) were added to the culture media. The aforementioned cells were maintained at 37°C and grown in a humidified incubator with 5% CO2 atmosphere.
Three siRNAs against POU6F2-AS2 (si-POU6F2-AS2) expression, NC siRNA (si-NC), E2F3 siRNA (si-E2F3), POU6F2-AS2 overexpression vector (pc-POU6F2-AS2), and E2F3 overexpression vector pcDNA3.1-E2F3 (pc-E2F3), were obtained from GenePharma Company (Shanghai, China). miR-125b-5p mimic, miRNA NC mimic (miR-NC), miR-125b-5p inhibitor, and NC inhibitor were obtained from RiboBio Co., Ltd. (Guangzhou, China). Transient transfection was performed using Lipofectamine 2000 (Invitrogen).
Three siRNAs against POU6F2-AS2 (si-POU6F2-AS2) expression, NC siRNA (si-NC), E2F3 siRNA (si-E2F3), POU6F2-AS2 overexpression vector (pc-POU6F2-AS2), and E2F3 overexpression vector pcDNA3.1-E2F3 (pc-E2F3), were obtained from GenePharma Company (Shanghai, China). miR-125b-5p mimic, miRNA NC mimic (miR-NC), miR-125b-5p inhibitor, and NC inhibitor were obtained from RiboBio Co., Ltd. (Guangzhou, China). Transient transfection was performed using Lipofectamine 2000 (Invitrogen).
7
Nasal Polyp Epithelial Cell Stimulation
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Biopsy specimens were collected from nasal polyps of CRSwNP and in vitro exposed to different stimuli, compared to epithelial cells derived from ethmoid tissue of CRSsNP. All specimens were immediately placed in BEGM medium (Bronchial Epithelial Cell Growth Media, Clonetics-Lonza) with no serum but supplemented with penicillin 100 U/ml, streptomycin 100 µg/ml, and glutamine.
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