We obtained primary HBE cells from Lonza (CC–2540S or CC–2540 for healthy donors, 00196979 for the CF donor). They were cultured in T–25 flasks using PneumaCult–Ex Plus medium (Stemcell Technologies) for no more than 3 passages. When they reached confluence, the cells were detached from the flask using the Animal Component–Free Cell Dissociation Kit (Stemcell Technologies) and centrifuged before being resuspended in PneumaCult–Ex Plus to a density of approximately 20,000 cells/μl.
Cc 2540
Manufactured by Lonza
Sourced in United States
The CC-2540 is a specialized laboratory equipment designed for cell culture applications. It serves as a reliable incubator for maintaining optimal environmental conditions for cell growth and proliferation. The core function of the CC-2540 is to provide a controlled temperature, humidity, and gas composition within the incubation chamber.
Automatically generated - may contain errors
Lab products found in correlation
Bronchial epithelial growth medium (begm), by Lonza (5 mentions)
Cc 3171, by Lonza (5 mentions)
Cc 3170, by Lonza (5 mentions)
Singlequots kit, by Lonza (3 mentions)
Singlequots kit, by Thermo Fisher Scientific (3 mentions)
Begm bullet kit, by Lonza (3 mentions)
Bronchial epithelial growth kit, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Evom2, by World Precision Instruments (2 mentions)
Fungizone, by Thermo Fisher Scientific (2 mentions)
Hela h1, by American Type Culture Collection (2 mentions)
Hek293t crl 3216, by American Type Culture Collection (2 mentions)
Rd ccl 136, by American Type Culture Collection (2 mentions)
C6 36 cells, by American Type Culture Collection (2 mentions)
293ft cells r70007, by Thermo Fisher Scientific (2 mentions)
Htb 37, by American Type Culture Collection (2 mentions)
Htb 55, by American Type Culture Collection (2 mentions)
Pneumacult ex plus, by STEMCELL (1 mentions)
Animal component free cell dissociation kit, by STEMCELL (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
37 protocols using cc 2540
1
Culturing Primary Human Bronchial Epithelial Cells
2
Culture of Bronchial Epithelial Cells
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The human bronchial epithelial cell line (BEAS-2B) was kindly provided by the Biomedical Research Institute at Seoul National University Hospital. BEAS-2B cells were maintained in defined keratinocyte serum-free medium containing epidermal growth factor, 100 U ml−1 penicillin, and 100 μg ml−1 streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). Primary NHBE cells (CC-2540, Lonza Group, Allendale, NJ, USA) isolated from the epithelial lining of airways above the bifurcation of a normal human donor lung were cultured in Bronchial Epithelial Growth Medium (BEGM BulletKit medium, CC-3171, Lonza) and used before passage 3 in all experiments. Cell cultures were incubated at 37 °C in humidified atmosphere containing 5% CO2.
3
Differentiation of Primary Human Bronchial Epithelial Cells
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Cryopreserved, primary normal human bronchial epithelial (NHBE) cells from one donor were purchased from Lonza (CC2540, lot 307177, 40Y Female; Walkersville, MD). Donor had no history of smoking or preexisting lung conditions. The subjects cause of death and presence of nonlung-related diseases remains unknown. Cryopreserved cell stock was expanded in bronchial epithelial growth medium (BEGM, Lonza) at a density of 3500 cells/cm2. Cells were frozen down at passage 2, expanded, and seeded onto human collagen-IV (Sigma, St. Louis, MO) coated 6.5 mm Transwell inserts (Costar, Corning, NY) at a density of 3.0 × 105 cells/cm². Cultures were maintained for 3 days in immersed conditions until confluency was reached. An air–liquid interface (ALI) was established by removing the apical medium and incubating with differentiation medium in the basolateral compartment only. Differentiation medium was comprised of a 50:50 mixture of LHC-basal medium (Gibco, Carlsbad, CA) and DMEM-H (Gibco) and supplemented as previously described by Fulcher et al. (2005 (link)). Cells were maintained at an air–liquid interface for up to 3 weeks, with differentiation media replenished every other day.
4
Isolation and Maintenance of NHBE Cells
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Normal human bronchial epithelial (NHBE) cells (Lonza, CC-2540 Lot# 580580), isolated from a 79-year-old Caucasian female and were maintained at 37 °C and 5% CO2 in bronchial epithelial growth medium (Lonza, CC-3171) supplemented with SingleQuots (Lonza, CC-4175) as per manufacturer’s instructions. Cells were maintained at the BSL3 facilities of the Icahn School of Medicine at Mount Sinai. NHBE cells (ATCC, PCS-300–010 Lot#63979089; #70002486), isolated from a 69-year-old Caucasian male and a 14-year-old Hispanic male were maintained in airway epithelial cell basal medium (ATCC, PCS-300–030) supplemented with Bronchial Epithelial Growth Kit as per the manufacturer’s instructions (ATCC, PCS-300–040) at 37 °C and 5% CO2. Cells were maintained at the BSL2 facilities of The Hebrew University of Jerusalem and the BSL3 facility of the central virology laboratory of the ministry of health and Sheba Medical Center.
Cells were authenticated at the source and routinely screened for mycoplasma using PCR.
Cells were authenticated at the source and routinely screened for mycoplasma using PCR.
5
Bronchial Epithelial Cell Culture and Calcium Regulation
6
Generation of Bronchial Organoids and ALI Model
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Bronchial organoids (BOs) were generated as described previously25 . Briefly, to generate BOs, NHBEs (Lonza, CC-2540) were suspended in 10 mg ml−1 cold Matrigel growth factor reduced basement membrane matrix and then cultured with the differentiation medium for 10 days. To generate a BO-derived air–liquid interface (ALI) model, expanding BOs were seeded into Transwell inserts (Corning) in a 24-well plate (3 × 104 cells per well) and then cultured with the differentiation medium for 5 days. The medium was only placed into the bottom chamber to maintain the ALI culture condition.
7
Primary Bronchial Epithelial Cell Culture
8
Cell Culture and Heavy Metal Exposure
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H460 and HBEC3-KT cells were obtained
from the American Type Culture Collection (ATCC). H460 cells were
grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v)
fetal bovine serum (FBS), and penicillin/streptomycin. HBEC3-KT cells
were propagated in Airway Epithelial Cell Basal Medium (PCS-300-030,
ATCC) with added Bronchial Epithelial Growth Kit (PCS-300-040, ATCC).
Primary human bronchial epithelial cells were obtained from Lonza
and propagated in the vendor’s recommended serum-free medium
(BEBM, CC-2540) supplemented with growth factors (CC-3170, Lonza).
All cell lines were grown in the atmosphere of 95% air/5% CO2. Other cell culture media tested for metals were DMEM (Gibco, 12430-062),
F12-K (ATCC, 30-2004), and EMEM (ATCC, 30-2003). Cells were treated
with the indicated concentrations of Cr(VI) next day after seeding.
Stock solutions of K2CrO4 (in water), NiCl2 (in water), MnCl2 (in water), iron(III) citrate
(in water), and iron(III) chloride (in 10 mM HCl) were freshly prepared
for each experiment.
from the American Type Culture Collection (ATCC). H460 cells were
grown in RPMI-1640 media (22400089, ThermoFisher) containing 10% (v/v)
fetal bovine serum (FBS), and penicillin/streptomycin. HBEC3-KT cells
were propagated in Airway Epithelial Cell Basal Medium (PCS-300-030,
ATCC) with added Bronchial Epithelial Growth Kit (PCS-300-040, ATCC).
Primary human bronchial epithelial cells were obtained from Lonza
and propagated in the vendor’s recommended serum-free medium
(BEBM, CC-2540) supplemented with growth factors (CC-3170, Lonza).
All cell lines were grown in the atmosphere of 95% air/5% CO2. Other cell culture media tested for metals were DMEM (Gibco, 12430-062),
F12-K (ATCC, 30-2004), and EMEM (ATCC, 30-2003). Cells were treated
with the indicated concentrations of Cr(VI) next day after seeding.
Stock solutions of K2CrO4 (in water), NiCl2 (in water), MnCl2 (in water), iron(III) citrate
(in water), and iron(III) chloride (in 10 mM HCl) were freshly prepared
for each experiment.
9
Differentiation of Normal Human Bronchial Epithelial Cells
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Cryopreserved normal human bronchial epithelial (NHBE; CC-2540, donor 1: TAN 24717, Lot No. 000312626; donor 2: TAN 36585, Batch: 18TL269120) cells were obtained from Lonza, and undifferentiated cells were seeded on collagen-coated transwell plates (Corning Costar, Corning, NY, USA). Cells were grown in a mixture of DMEM and bronchial epithelial cell growth medium (BEGM, Lonza, Basel, Switzerland) supplemented with retinoic acid (75 nM, Sigma Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 atmosphere. Fresh medium was added regularly after 2 days. After reaching confluence, the cells were cultivated under air-liquid interface conditions for 4 additional weeks for full differentiation into pseudostratified human airway epithelia. Medium from the basolateral compartment was replaced with fresh medium every 2–3 days, and the apical surface was washed every week with PBS (Invitrogen, Carlsbad, CA, USA).
10
Culturing Human and Canine Cell Lines
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Human embryonic kidney 293T (HEK293) cells and Madin-Darby canine kidney (MDCK; NBL-2) cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Corning antibiotic-antimycotic solution) at 37°C and 5% CO2. Normal human bronchial epithelial (NHBE) cells (Lonza; CC-2540, lot no. 630564) were isolated from a 16-year-old Caucasian female and were differentiated in an air-liquid interface following the manufacturer’s instructions (Lonza; CC-4175) at 37°C and 5% CO2.
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