BECs were isolated from large bronchial airway of rat as described in the literature [26 (link)]. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the Tongji Medical College, Huazhong University of Science and Technology. In brief, airway was isolated from rat under sterile conditions, rinsed with ice D-Hanks twice, then digested with 1% Pronase E in DMEM/F-12 at 4°C for 14 h. Then BECs were harvested with ice D-hanks contain 5% newborn bovine serum (NBS) and centrifuged at 1000 rpm for 5 min. The spun down cells were resuspended with epithelial cell medium (Cell Biologics, Chicago, IL, USA) and incubated in a 5% CO2 incubator (Thermo Fisher Forma, Waltham, MA, USA) at 37 °C. These cells were used in experiments without subculture. In most experiments, BECs were treated by the indicated factors in serum-free medium when the cells were 60–70% confluence. For MUC5AC immunostaining, the isolated cells digested with protease were cultured on cover slip with serum-containing medium for 24 h in the Petri dish. Some digested cells were presented as small clusters, but not completely separated cells. IL-4 containing serum-free medium was administered when the cells reached about 30% confluence.
Epithelial cell medium
Manufactured by Cell Biologics
Sourced in United States
Epithelial cell medium is a specialized cell culture medium designed to support the growth and maintenance of epithelial cells. It provides the necessary nutrients, growth factors, and other components required for the optimal proliferation and differentiation of epithelial cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Primary small iecs, by PELOBiotech (2 mentions)
Gelatin based coating solution, by Cell Biologics (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
P stat6, by Cell Signaling Technology (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Anti p65 antibody, by Santa Cruz Biotechnology (1 mentions)
Ikk16, by MedChemExpress (1 mentions)
As1517499, by MedChemExpress (1 mentions)
Anti caveolin 1 antibody, by BD (1 mentions)
Pronase e, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fura 2 am, by Dojindo Laboratories (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Clioquinol, by Merck Group (1 mentions)
Disulfiram, by Merck Group (1 mentions)
T75 flask, by Avantor (1 mentions)
Fetal bovine serum (fbs), by Cell Biologics (1 mentions)
Co2 humidified incubator, by Thermo Fisher Scientific (1 mentions)
7 protocols using epithelial cell medium
1
Isolation and Culture of Rat Bronchial Epithelial Cells
2
Isolation of Rat Pleural Primary Mesothelial Cells
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Primary PMCs were isolated from rat pleura with pronase E digestion. In brief, the rat was anesthetized by intraperitoneal injection with 7% chloral hydrate (5 μl/g body wt) and then 5 ml 1% pronase E in RPMI-1640 were injected into thoracic cavity. The rat was then killed by using carbon dioxide. The whole thorax was isolated by cutting the lumbar vertebrae, cervical vertebrae, and connected tissues under sterile conditions, and the pleura was digested by pronase E at 4°C overnight. PMCs were harvested from thoracic cavity and centrifuged at 1,000 rpm for 5 min. The spun down cells were resuspended with epithelial cell medium (Cell Biologics, Chicago, IL) and cultured in a 5% CO2 incubator at 37°C.
3
Culturing Primary Small Intestinal Epithelial Cells
4
Culturing Primary Small Intestinal Epithelial Cells
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Primary small IECs were purchased from PELOBiotech. Cells were cultivated in Epithelial Cell Medium (Cell Biologics) in T25 flasks pretreated with gelatin-based coating solution (Cell Biologics). During expansion, medium was replenished and cells were split as needed.
5
Molecular Mechanisms of Airway Inflammation
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Methyl-β-cyclodextrin (M-βCD), 2-Aminoethyl diphenylborinate ( 2-APB), ethylene glycol tetraacetic acid (EGTA) and Pronase E were purchased from Sigma-Aldrich (St. Louis, MO, USA). Epithelial cell medium was obtained from Cell Biologics (Chicago, IL, USA). IL-4 was obtained from Pepro Tech (Rocky Hill, NJ, USA). Anti-caveolin-1 antibody was purchased from BD Transduction Laboratories (Lexington, KY, USA), rabbit anti-TRPC1 polyclonal antibody was purchased from Abcam (Cambridge, UK), anti-P65 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cholera toxin subunit B-Alexa Fluor 488 and Trizol Reagent were obtained from Life Technology (Grand Island, NY, USA). Fura-2-AM was purchased from Dojindo laboratories (Kumamoto, Japan). MUC5AC ELISA kit was obtained from Cloud-Clone Corp (Wuhan, China). Anti-MUC5AC antibody was purchased from Absin (Shanghai, China). Antibodies against β-actin, p-STAT6 and p-P65 were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific STAT6 inhibitor AS1517499 and selective IκB kinase (IKK) inhibitor IKK16 was purchased from MedChem Express (Monmouth Junction, NJ, USA).
6
Copper-Ionophore Treatment of Prostate Cancer
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TRAMP-C1 mouse prostate cancer cells were derived from the TRAMP (transgenic adenocarcinoma of mouse prostate) strain (C57BL/6 background) [38 (link)] and were generously provided by Assoc. Prof. Michael H. Kershaw (Peter MacCallum Cancer Centre, Melbourne, Australia). Mouse (C57BL/6) primary prostate epithelial cells (PrECs) were purchased from Cell Biologics (Chicago, USA; Cat#C57-6038). TRAMP-C1 cells were cultured in DMEM (ThermoFisher, Scoresby, Australia; Cat#11965-092) supplemented with 10% foetal calf serum (Bovogen biologicals, Keilor East, Australia; Cat#SFBS-F), 2 mM L-glutamine, 100 Units/mL penicillin and 100 μg/mL streptomycin. Primary prostate epithelial cells were cultured in Epithelial Cell Medium (Cell Biologics, Chicago, USA; Cat# M6621) as per the manufacturer's instructions. Cells were maintained at 37°C under humidified atmosphere containing 5% CO2.
Copper-ionophores (Disulfiram; Cat#86720 and clioquinol; Cat#24880) were purchased from Sigma-Aldrich (Castle Hill, Australia) or synthesized [CuII(gtsm)] by Assoc. Prof. Paul S. Donnelly (University of Melbourne, Melbourne, Australia) following published procedures [70 ]. Each copper-ionophore was prepared in DMSO at 5 mM immediately before each experiment. All other reagents were supplied by Sigma-Aldrich (Castle Hill, Australia) unless specified otherwise.
Copper-ionophores (Disulfiram; Cat#86720 and clioquinol; Cat#24880) were purchased from Sigma-Aldrich (Castle Hill, Australia) or synthesized [CuII(gtsm)] by Assoc. Prof. Paul S. Donnelly (University of Melbourne, Melbourne, Australia) following published procedures [70 ]. Each copper-ionophore was prepared in DMSO at 5 mM immediately before each experiment. All other reagents were supplied by Sigma-Aldrich (Castle Hill, Australia) unless specified otherwise.
7
Isolation and Culture of Mouse Alveolar Epithelial Cells
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C57BL/6 mouse primary alveolar epithelial cells were isolated from lung tissue of pathogen-free laboratory mice (Cell Biologics). Cells were cultured in an epithelial cell medium (Cell Biologics) with growth factors (0.01% insulin-transferrin-selenium, 0.01% EGF, 1% L-glutamine, 1% antibiotic-antimycotic solution, 10% FBS, Cell Biologics, Chicago, IL, USA). Cells were cultured in T75 flasks (VWR) in a 5% CO2 humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C.
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