BEAS-2B所用培养基.doc

1、 BEBM培养基（ATCC）(1个)1.African Dust Storms Reaching Puerto Rican Coast Stimulate the Secretion of IL-6 and IL-8 and Cause Cytotoxicity to Human Bronchial Epithelial Cells (BEAS-2B)BEAS-2B cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Cat No CRL-9609). Cells were cultured according to ATCC protocols, maintained in keratinocyte basal medium (KBM-2, Lonza, Walkersville, MD, USA Cat No CC 3103) and supplemented with keratinocyte growth medium (KGM-2 SingleQuots; Lonza, Walkersville, MD, USA; Cat No CC4152). Cells were used at passages 44 - 59 and maintained at 37C in a humidified atmosphere of 5% CO2 2、 DMEM（11）1.Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal CellsIn an independent experiment, A549 cells (2103 cells well-1) were seeded in a 96-well plate and kept overnight for attachment. The next day the medium was replaced with fresh medium with three concentrations (2, 5 and 10 M) for each of three PUAs (DD, OD, and HD, Sigma-Aldrich Inc., Milano, Italy) tested and with caspase-3 Inhibitor (C30H41FN4O12, sc-3075, Santa Cruz) at 9.7 M; cells were allowed to grow for 24, 48 and 72 h. After aldehyde treatment, viable cells were evaluated as described below. The BEAS-2B (ATCC CRL-9609) lung/brunch normal epithelial cell line was maintained in DMEM (Dulbeccos modified Eagles medium) supplemented with 50% fetal bovine serum (FBS), 100 units ml1 penicillin and 100 g ml1 streptomycin. Cells were incubated in a 5% CO2 humidified chamber at 37C for growth. BEAS-2B (2103 cells well1) was seeded in a 96-well plate and kept overnight for attachment. The next day the medium was replaced with fresh medium with three concentrations (2, 5 and 10 M) for each of three PUAs (DD, OD, and HD, Sigma-Aldrich Inc., Milano, Italy) tested; cells were allowed to grow for 24, 48 and 72 h. After incubation, the supernatant was removed and adherent cells were examined for viability.2.Gene Expression Profile and Toxic Effects in Human Bronchial Epithelial Cells Exposed to ZearalenoneHuman bronchial epithelial BEAS-2B cell line 19 (from the American Type Culture Collection, ATCC) was cultured in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml of penicillin and 10 g/ml of streptomycin. All cells were maintained in a 37C humidified incubator with 5% CO2. DMEM with Geneticin (G418, 200 g/ml) were used to maintain and select Cygb overexpressing cells.3.Arsenic-induced sub-lethal stress reprograms human bronchial epithelial cells to CD61 cancer stem cellsThe human bronchial epithelial cell line BEAS-2B was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM with 10% FBS. For As3+ treatment, BEAS-2B cells were treated with 0.25 M NaAsO2 continuously for six months. Cells cultured without As3+ treatment for six months were used as parental cell control in all experiments. Cell transformation was evaluated by using in vitro soft agar colony-forming assay and in vivo tumorigenicity experiment in athymic nude mice. For the soft agar colony-forming assay, 1% agar was melted and mixed with 2 DMEM medium in a 1:1 ratio to produce a supporting layer (0.5%) in a 6-well tissue culture plate (1 ml/well). The bottom agar layer was allowed to solidify at room temperature for 20 min. The top layer containing 0.3% agar (1 ml/well) was prepared by mixing stock agar solutions with 5,000 cells in 2 DMEM medium and was laid on the top of the bottom agar layer. Fresh DMEM medium was added every 3 days. Three weeks later, colonies were stained with a 0.05% crystal violet solution, counted, and photographed under a microscope4.Receptor Interacting Protein-2 Plays a Critical Role in Human Lung Epithelial Cells Survival in Response to Fas-Induced Cell-DeathHuman lung epithelial cells (BEAS-2B) were purchased from ATCC (ATCC CRL9609). BEAS-2B cells were cultured in DMEM (MediaTech, Inc) supplemented with 10% heat-inactivated FBS (Atlanta Biologicals) and 1% penicillin-streptomycin (Invitrogen Life Technologies). Anti-Fas (human, activating), clone CH11 was purchased from Upstate Cell Signaling Solution, USA. Cells were regularly checked to confirm the absence of Mycoplasmacontamination 16. Cell culture medium was used for detection of LDH release as a cell death signature. Cell lysates were analyzed for proteins by immunoblots.5.NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human CellsHuman BEAS-2B lung epithelial cells were from the American Type Culture Collection (ATCC, Manassas, VA). Human pulmonary fibroblasts (hPF, ScienCell, Carlsbad, CA) and human embryonic kidney 293 (HEK293) cells were grown at ambient oxygen levels and 10% CO2 in DMEM supplemented with 10% fetal calf serum before treatment. Our laboratories have been using HEK293 cells for many transfection-based experiments, therefore, this cell line was used along with the two pulmonary cell lines. Cells were treated with various concentrations of SSS for 24 h before collecting by trypsinization.6.MicroRNA-34c is associated with emphysema severity and modulates SERPINE1 expression BEAS-2B and HFL1 were cultured in RPMI and DMEM, respectively, supplemented with antibiotics and 10% FCS and incubated in 5% CO2.7.Chronic Arsenic Exposure and Angiogenesis in Human Bronchial Epithelial Cells via the ROS/miR-199a-5p/HIF-1/COX-2 PathwayCell culture and generation of stable cell lines. Human bronchial epithelial BEAS-2B cells American Type Culture Collection (ATCC), Manassas, VA, USA were cultured in DMEM (Dulbeccos modied Eagles medium; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS).8. Differential responses to genotoxic agents between induced pluripotent stem cells and tumor cell linesMitomycin C treated murine embryonic fibroblasts (MEFs) were prepared as feeder cells. Tera-1 cells obtained from American Type Culture Collection (ATCC) were cultured in McCoys 5A medium supplemented with 10% fetal bovine serum (FBS). BEAS-2B cells obtained from ATCC were cultured in DMEM supplemented with 10% FBS.9The effect of particle size, location and season on the toxicity of urban and rural particulate matterthe bronchial epithelial cell line (BEAS-2B) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in DMEM medium (Dulbeccos Modified Eagle Medium; Gibco) with 10% FBS (Gemini Bio Produ10.Perilla frutescens Leaf Extract Inhibits Mite Major Allergen Der p 2-induced Gene Expression of Pro-Allergic and Pro-Inflammatory Cytokines in Human Bronchial Epithelial Cell BEAS-2BThe nontumorigenic human bronchial epithelial cell BEAS-2B (ATCC CRL-2503) was obtained from American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in Dulbeccos modified Eagles medium (DMEM) containing 10% v/v fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) and 100 g/mL penicillin/streptomycin (Sigma, St. Louis, MO) at 37C in a humidified atmosphere containing 5% CO2.11.Sodium metavanadate exhibits carcinogenic tendencies in vitroin immortalized human bronchial epithelial cellsImmortalized human bronchial epithelial cells (Beas-2B; #CRL-9609, ATCC, Manassas, VA) were cultured in 1 Dulbeccos Modified Eagle Medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 100 ug mL1 Pen Strep (GIBCO, Grand Island, NY) (“complete culture media”). The cells were maintained in 25 cm2 polystyrene tissue culture flasks in an incubator at 37C with 5% CO2 and 100% humidity. The media were changed every 2 days, and the cells were passaged every 4 days using 0.05% trypsin-EDTA (GIBCO).3、 F12 （1）1.Biological responses according to the shape and size of carbon nanotubes in BEAS-2B and MESO-1 cells BEAS-2B cells were cultured in Hams Nutrient Mixture F-12 (Nacalai Tesque) with 10% fetal bovine serum (FBS Life Technologies, Carlsbad, CA, USA), and MESO-1 cells were cultured in RPMI1640 supplemented with 10% FBS. 4、 DMEMF12 (3)1.Effect of Tumor Necrosis Factor Family Member LIGHT (TNFSF14) on the Activation of Basophils and Eosinophils Interacting with Bronchial Epithelial CellsBEAS-2B cells were grown in Dulbeccos modified Eagles medium nutrient mixture F12 (Invitrogen) with 10% FBS at 37C in a humidified 5% CO2 atmosphere until confluence to cell monolayer. In coculture, the medium of BEAS-2B cells was replaced with RPMI-1640 medium containing 10% FBS (Invitrogen) with or without basophils/eosinophils. For inhibition experiments, basophils/eosinophils and BEAS-2B cells were pretreated with signaling molecule inhibitors for 1h before coculture and treatment by LIGHT.2.Sphingosine 1-Phosphate (S1P) Induced Interleukin-8 (IL-8) Release Is Mediated by S1P Receptor 2 and Nuclear Factor B in BEAS-2B CellsHuman BEAS-2B cells (ATCC, Manassas, VA) were grown in DMEM:F12 10%FBS 100 U/ml penicillin, 100 g/ml streptomycin and 2500 ng/ml amphotericin B (PSA) (Invitrogen, Carlsbad, CA, USA) in 75 cm2 tissue culture flasks at 37C and 5% CO2. Culture medium was changed every 2 days and cells were seeded into new flasks when approximately 80% confluent. Cells were detached by incubation with 0.25% trypsin (Sigma-Aldrich). For experimentation, cells were seeded in 6 well plates at a density of 50 000 cells per well and grown for 3 days in culture medium. Cells were serum-starved for 24 hours in DMEM:F12 0.1%BSA (Sigma-Aldrich) with PSA. Starvation medium was changed prior to all experiments and culture supernatant was collected at the end of the incubation period.3.EFFECTS OF COREXIT DISPERSANTS ON CYTOTOXICITY PARAMETERS IN A CULTURED HUMAN BRONCHIAL AIRWAY CELLS, BEAS-2BCommercially available COREXIT 9500A, 9527A, and 9580A dispersants as liquid solutions were provided by a contract between Nalco/Exxon Energy Chemicals, L.P. (Sugar Land, TX) and Tulane University (New Orleans, LA). Fibronectin, collagen, Dulbeccos modified Eagles medium with nutrient mixture (DMEM/F-12), and penicillin/streptomycin were purchased from Sigma-Aldrich (St. Louis, MO). 五、LHC-9（中科院） (6)1.Transforming growth factor- impairs glucocorticoid activity in the A549 lung adenocarcinoma cell line BEAS-2B cells (from ATCC, Manassas, VA, USA) were cultured in LHC-9 media containing 2% vv1 heat-inactivated FCS, 15 mM HEPES, 0.2% vv1 sodium bicarbonate, 2 mM L-glutamine, 50 IUmL1 penicillin and 50 gmL1 streptomycin and maintained at 37C in a humidified 5% CO2/95% air atmosphere. For experiments, the cells were seeded at 25 000 cells/well in 24 well plates and incubated in serum-free medium (phenol red-free Roswell Park Memorial Institute (RPMI) containing 15 mM HEPES, 0.2% vv1 sodium bicarbonate, 50 IUmL1 penicillin, 50 gmL1streptomycin, 0.25% wv1 BSA, 2 mM L-glutamine, 1% v/v sodium pyruvate and 1% v/v non-essential amino acids) for 24 h prior to treatment with TGF- (40 pM) for 24 h. The cells were then treated with dexamethasone. IL-1 (1 ngmL1) was added 30 min after the addition of dexamethasone to stimulate the production of cytokines when required. Cells were supplemented with Monomed A (1% vv1) 24 h after culture in serum-free medium.2.Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-1A549 lung adenocarcinoma and BEAS-2B bronchial epithelial cell lines were cultured as described 9【9就是上面的文章】3.Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cellsThe cytotoxicity of cobalt oxide particles was evaluated on BEAS-2B cells by measuring the CellTiter-Glo assay after 72 h exposure to increasing concentrations of Co3O4 particles in LHC9 (01000 g.mL1 Co). Because of the low level of toxicity of cobalt oxide particles 26, we investigated a wide range of concentrations. In parallel, the cytotoxicity of LB-3 latex beads was evaluated for similar concentrations (01000 g.mL1). After 72 h exposure, the sedimentation of 400 nm cobalt oxide particles, as well as latex beads, was fully achieved which validate the cytotoxicity results obtained for these particles suspensions, all the particles being available to the adhering cells. 4.Curcumin and Vitamin E Protect against Adverse Effects of Benzoapyrene in Lung Epithelial CellsHuman bronchial epithelial cell line, BEAS-2B, was obtained from American Type Culture Collection (ATCC). The cells were cultured as described in our previous study 31. In brief, after the flasks/dishes/plates were coated, BEAS-2B cells were cultured in LHC-9 (Life Technology, MI) completed growth medium containing 50U penicillin/mL and 50 g/mL streptomycin. The cells were then incubated at 37C in 95% air and 5% CO2. At 80% confluence, cells were harvested using 0.05% trypsin/EDTA solution (HyClone) and were then sub-cultured.5.6. Regulation of CYP3A genes by glucocorticoids in human lung cellsBEAS-2B cells (American Type Culture Collection) were cultured in LHC-9 medium (Life Technologies). Lobar cells (donor number 01334) were cultured in BronchiaLife Basal Medium supplemented with the BronchiaLife B/T supplement kit (Lifeline Cell Technology, Walkersville, MD). 六、BEGM (2)1.2.Brd4 Is Essential for IL-1-Induced Inflammation in Human Airway Epithelial CellsNormal human bronchial epithelial cells (NHBE) of non-smoking subjects were obtained from Lonza (Lonza, Slough, UK) and grown in br