The immortalized human bronchial epithelial cell line, BEAS-2B (European Collection of Cell Cultures) was cultured in PneumaCult™-Ex Plus Medium (Stemcell Technologies, United Kingdom) supplied with 50x extra supplement; hydrocortisone (Stemcell Technologies, United Kingdom) and penicillin-streptomycin solution (Gibco, Sweden) was added to the complete cell medium. It is important to note that the cell medium is free from FBS and bovine pituitary extract (BPE). Hence, the cell culture medium can be considered “animal-free” (Oredsson et al., 2019 (link)). Furthermore, BEAS-2B cells are often grown on a substrate of fibronectin, collagen, and serum albumin of bovine origin. However, we were able to maintain cells without pre-coating with extracellular matrix proteins (Supporting Information), thus avoiding the use of animal proteins. Hence, the cells were seeded in 75 cm2 tissue culture flasks without pre-coating and expanded until 70–80% confluence for further studies under static or dynamic conditions, as described below.
Beas 2b
Manufactured by European Collection of Cell Cultures
Sourced in United Kingdom
BEAS-2B is a human bronchial epithelial cell line derived from normal human bronchial epithelial cells. It is commonly used in research related to respiratory and lung biology.
Automatically generated - may contain errors
Lab products found in correlation
Pneumacult ex plus medium, by STEMCELL (2 mentions)
Hydrocortisone, by STEMCELL (2 mentions)
Bronchial epithelial growth medium (begm), by Lonza (2 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Beas 2b, by Lonza (1 mentions)
Begm singlequots kit, by Lonza (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Lhc 9 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by MP Biomedicals (1 mentions)
8 protocols using beas 2b
1
Animal-Free Culture of BEAS-2B Cells
2
Culturing BEAS-2B Bronchial Epithelial Cells
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The normal bronchial epithelial cell line (BEAS-2B, European Collection of Cell Cultures) was used in this study. BEAS-2B cells were cultured in Bronchial epithelial cell growth medium (BEGM™, Lonza) supplemented with recombinant epidermal growth factor (EGF), hydrocortisone, insulin, bovine pituitary extract, GA-1000 (Gentamicin Sulfate and Amphotericin-B), retinoic acid, transferrin, triiodothyronine, epinephrine according to manufacturers’ instructions. No fetal calf serum was added in the cell medium. The cells were seeded in flasks and plates pre-coated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I, 0.01 mg/mL bovine serum albumin and 0.2% penicillin-streptomycin in BEGM additive free medium. The cells were incubated in a humidified atmosphere at 37°C, 5% CO2 and sub-cultured at 80% confluency.
For each experiment, BEAS-2B cells were seeded one day prior to AgNPs exposure, at an approximate density of 3 × 104 cells/cm2 for 24 h exposure and 6 × 104 cells/cm2 for 4 h exposure in suitable cell culture plates.
For each experiment, BEAS-2B cells were seeded one day prior to AgNPs exposure, at an approximate density of 3 × 104 cells/cm2 for 24 h exposure and 6 × 104 cells/cm2 for 4 h exposure in suitable cell culture plates.
3
BEAS-2B Cell Culture and Exposure
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The immortalised human bronchial epithelial cell lines (BEAS-2B, European Collection of Cell Cultures) were cultured in bronchial epithelial cell growth medium (BEGM; Lonza) supplemented with human epidermal growth factors (hydrocortisone, insulin, bovine pituitary extract, gentamicin and amphotericin-B, transferrin, triiodothyronine, retinoic acid and epinephrine) according to manufacturer’s instructions (BEGM SingleQuots Kit, Lonza). The cells were seeded in flasks or multi-well plates pre-coated with a mixture of 0.01mg/ml fibronectin, 0.03mg/ml bovine collagen type I, 0.01mg/ml BSA and 0.2% penicillin–streptomycin in BEGM supplement-free medium. The cells were incubated in a humidified atmosphere at 37°C, 5% CO2 and subcultured at 80% confluency for no more than 10 passages. For each experiment, BEAS-2B cells were seeded one day prior to particles exposures, at an approximate density of 6×104 cells/cm2 for 24- and 48-h exposure and 15×104 cells/cm2 for 3-h exposure in indicated cell culture plates. The final exposure volumes used in different plates were determined in order to keep the same µg/cm2 correspondence between exposures in different plates.
4
Lung and Breast Cancer Cell Lines
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Eight lung cancer cell lines (A549, Calu‐3, HCC827, NCI‐H1299, NCI‐H1781, NCI‐H1975, NCI‐H1993, and NCI‐H2170), two breast cancer cell lines (SK‐BR‐3 and BT‐474), and one normal human bronchial epithelial cell line (BEAS‐2B) were used in this study. These cell lines, except A549, SK‐BR‐3, BT‐474 and BEAS‐2B, were kindly provided by Dr. Adi F. Gazdar (The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA), who established these lines with Dr. John D. Minna.19 , 20 A549, SK‐BR‐3, and BT‐474 were purchased from ATCC (Manassas, VA, USA). BEAS‐2B was purchased from European Collection of Cell Cultures (Porton Down, UK). All lung cancer cells were cultured in RPMI‐1640 media supplemented with 10% FBS and the other cells were maintained in DMEM with 10% FBS. They were grown in a humidified incubator with 5% CO2 at 37°C. Afatinib and gefitinib were purchased from Sykkinase (San Diego, CA, USA) and InvivoGen (San Diego, CA, USA), respectively.
5
BEAS-2B Cell Culture and Cytotoxicity Assays
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The immortalized human cell line BEAS-2B was obtained from European Collection of Cell Cultures. The cells were cultured in PneumaCult™-Ex Plus Medium (Stemcell Technologies, United Kingdom) supplied with 50x extra supplement; hydrocortisone (Stemcell Technologies), and penicillin-streptomycin (Gibco, Sweden) (Mukherjee et al., 2020 (link)). Cells were seeded at a density of 25,000 cells/cm2, and cytotoxicity was evaluated using the Alamar blue and LDH release assays, as described below.
6
Cultivation of A549 and BEAS-2B Cell Lines
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Human bronchial carcinoma cells A549 (DSMZ, Braunschweig, Germany; DSMZ no.: ACC 107) and the bronchial epithelial cell line BEAS-2B (European Collection of Cell Cultures, ECACC, Salisbury, UK; Catalogue No.: 95102433) were cultivated at 37 °C, 5% CO2 in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin. The cell lines were passaged once weekly to ensure exponential growth.
7
Airway Epithelial Cell Response to PM2.5
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The airway epithelial cell line BEAS-2B was purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, UK) and maintained by subculture in 37°C at 5% CO2 in LHC-9 medium. Cells were exposed to an aqueous or organic extract of PM2.5 at the concentrations of 0, 7.5, 22.5 or 75 µg/mL for 24 hr. We measured the cell viability and the secretion of the cytokines IL-6 and IL-8 from the airway epithelial cells after 24 hr of exposure to the aqueous or organic extract by conducting a Water Soluble Tetrazolium Salts (WST-1) assay and quantikine Enzyme Linked Immuno Sorbent Assay (ELISA), respectively.
8
Culturing Human Respiratory Epithelial Cells
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The RPMI-2650, derived from squamous cell carcinoma of nasal septum was used as model of human nasal epithelial cells which are cells of the upper respiratory tract. These cells display consistent growth and high stability throughout continued culturing in vitro with no alteration to the normal diploid karyotype (Moorhead, 1965) . The cell line was purchased from the European Collection of Cell Cultures (Salisbury, Wiltshire, United Kingdom) and maintained in Eagle's minimal essential medium (DS Pharma Biomedicals, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (MP Biomedicals, Eschwege, Germany), 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma, St Louis, Missouri). As representative of the cells of the lower respiratory tract, the BEAS-2B, derived from human bronchial epithelial cells, was purchased from the European Collection of Cell Cultures and maintained in LHC-9 medium (Thermo Scientific, Waltham, Massachusetts) which is serum-free medium containing Gentamicin. RPMI-2650 cells and BEAS-2B cells were maintained by subculture in 37°C at 5% CO2 in medium.
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