hBE33 cells are an immortalized human airway epithelial cell line derived from a male donor and engineered to stably express IL-33eGFP (73 (link)). hBE33 cells are maintained in BronchiaLife medium supplemented with grown factors and retinoic acid (Lifeline Cell Technology) as well as 1% penicillin/streptomycin. Once confluent, hBE33 cells were pretreated with 5α-DHT for 24 hours prior to exposure to Alt Ext (30 μg/mL) for 1 hour, and then basolateral supernatants were collected.
Bronchialife medium
Manufactured by Lifeline Cell Technology
Sourced in United States
BronchiaLife medium is a cell culture medium designed to support the growth and maintenance of human bronchial epithelial cells. It provides the necessary nutrients and growth factors to sustain these cells in an in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Retinoic acid, by Lifeline Cell Technology (1 mentions)
Penicillin and streptomycin ps solution, by Thermo Fisher Scientific (1 mentions)
Sagm medium, by Lonza (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Hsaecs, by PromoCell (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Complete human epithelial cell medium, by Cell Biologics (1 mentions)
Endo growth medium, by Neuromics (1 mentions)
Mcdb 131 medium, by Thermo Fisher Scientific (1 mentions)
Complete human endothelial cell medium, by Cell Biologics (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Gemini Bio (1 mentions)
Hulec 5a cells, by American Type Culture Collection (1 mentions)
Recombinant human il 13, by R&D Systems (1 mentions)
Pneumacult ali medium, by STEMCELL (1 mentions)
Rpmi 1640 medium, by Corning (1 mentions)
6 protocols using bronchialife medium
1
Assessing IL-33 Signaling in hBE33 Cells
2
Cultivation of Human Cell Lines for Research
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Human alveolar epithelial A549 cell line, HEK cells containing SV40 T‐antigen (HEK293T) and Madin–Darby canine kidney cell line (MDCK) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). A549 cells were cultured in F12K media containing 10% foetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA) and 0.1% penicillin and streptomycin (PS) solution (Life Technologies Corporation, Carlsbad, CA, USA). HEK293T and MDCK cells were cultured in DMEM media containing 10% FBS and 0.1% PS. dCas9‐ KRAB A549 stable cell line was previously generated10 and maintained in F12K containing 10% FBS, 0.1% PS and 1 μg/ml of puromycin. Human small airway epithelial cells (HSAECs) were purchased from PromoCell GmbH (Catalog # C‐12642, Sickingenstr, Heidelberg, DEU) and were cultured in SAGM medium (Catalog # CC‐3118, Lonza, Walkersville, MD, USA). Human bronchial epithelial cells (HBECs) were purchased from Lifeline Cell Technology (Catalog # FC‐0035, Frederick, Maryland, USA) and maintained in BronchiaLife™ medium (Lifeline Cell Technology, Catalog # LL‐0023).
3
Cultivation of Human Cell Lines
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HULEC-5a cells were obtained from the American Type Culture Collection (ATCC) and maintained in MCDB131 Medium (Gibco, Thermo Fisher) supplemented with 10ng/ml epidermal growth factor (Thermo Fisher), 1µg/ml hydrocortisone (Sigma Aldrich), 10mM L-glutamine (Thermo Fisher), and 10% (v/v) FCS (GeminiBio). Primary human small airway epithelial cells (HSAEC) were purchased (Lifeline Cell Technology) and maintained in BronchiaLife Medium (cat. no. LKL-0023, Lifeline Cell Technology). Primary human alveolar epithelial cells (HAEC) and primary human kidney glomerular endothelial cells (HKGEC) were purchased (CellBiologics) and maintained in Complete Human Epithelial Cell Medium (cat. no. H6621, CellBiologics) and Complete Human Endothelial Cell Medium (cat. no. H1168, CellBiologics), respectively. Primary human small intestine microvascular endothelial cells were purchased (Neuromics) and maintained in ENDO-Growth Medium (cat. no. EGK001, Neuromics). All cells were kept at 37°C in a humidified incubator supplemented with 5% CO2 and maintained between 50–80% confluence. Primary cells were grown in cell culture flasks coated with gelatin (cat. no. 6950, CellBiologics) and used between 3–7 passages.
4
Isolation and Differentiation of Primary HTBE Cells
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Detailed method on isolation of primary HTBE cells from normal healthy donors were described in our previous publication (26 (link)). Isolated HTBE cells were expanded on collagen-coated 100 mm dishes containing BronchiaLife medium with supplements (Lifeline Cell Technology, Frederick, MD, USA). Air–liquid interface (ALI) culture was performed by seeding the cells onto collagen-coated 12-well transwell plates (Transwell 2460, Corning Incorporated, Corning, New York, USA) with PneumaCult-ALI medium (StemCell, Vancouver, British Columbia, Canada). After 7 days of submerged culture, cells were shifted to ALI for the next 21 days to induce mucociliary differentiation. Cells were stimulated with 10 ng/ml recombinant human IL-13 (R&D Systems, Minneapolis, Minnesota, USA) for three consecutive days starting on day 21 of ALI. On days 22 and 23, cells were treated with 100 µM PA conjugated with 0.1% fatty acid-free BSA or 0.1% fatty acid-free BSA as a control. Cells, apical and basolateral supernatants were harvested on day 24 (24 h after the last IL-13/PA treatment). Cells were lysed with RIPA buffer for western blot analysis. Supernatants were used for the DPP4 activity assay and ELISA.
5
Culturing Human Bronchial and THP-1 Cells
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Human bronchial epithelial (16HBE) cells were cultured in BronchiaLife medium (Lifeline Cell Technology) supplemented with 1% FBS and 1% penicillin/streptomycin. THP-1 cells were grown in RPMI 1640 medium (Corning) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were incubated at 37°C in 5% CO2.
6
Air-Liquid Culture of Airway Basal Cells
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Bronchial tissue segments from normal lung donors and explanted lungs from COPD patients were collected at the time of lung transplantation. COPD patients provided written informed consent forms to use their explanted lungs for research (IRB# 4407). Donors of normal lungs had standing consent to use their organs for organ transplant and research after their demise. The collection and use of the tissue samples were approved by University of Michigan (IRB# HUM00050876) and Temple University (IRB# 23903) institutional review boards. All methods were performed in accordance with the relevant guidelines and regulations. Patient characteristics are provided in supplementary table S1 . Airway basal cells from bronchial segments were isolated and expanded in the BronchiaLife medium (Lifeline Cell Technology, Frederick, MD, USA). The basal cells at passage 1 were cultured in 12-mm transwells at an air/liquid interface to promote mucociliary differentiation, as described previously [10 (link), 19 (link)]. We seed ∼90 000 live cells·cm−2 in a transwell, and this seeding strategy did not indicate difference in growth rate between COPD and normal cells.
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