Primary normal human bronchial epithelial (NHBE) cells were obtained from LONZA Walkersville Inc. (NHBE CC-2540; Lonza) from a single anonymous female donor, who was a non-smoker with no respiratory pathology. NHBE cells were seeded and grown according to the manufacturer's instructions. Briefly, cells were passaged once into a T25 flask in BEBM Bronchial Epithelial Cell Growth Basal Medium (CC-3171, Lonza) with BEGM Bronchial Epithelial Cell Growth Medium SingleQuots Supplements and Growth Factors, containing Bovine Pituitary Extract [BPE], Hydrocortisone, human Epidermal Growth Factor [hEGF], Epinephrine, Transferrin, Insulin, Retinoic Acid, Triiodothyronine, and Gentamicin/Amphotericin-B (CC-4175, Lonza). The growth media was changed every 48–72 h. When cells exceeded 45% confluence, the volume of the medium was doubled. Once cells reach 75–85% confluence, cells were re-seeded at 100,000 cells/T25 flask. Cells were passaged every seven days or when 85% confluency was reached. We used Clonetics ReagentPack (CC-5034, Lonza) for cell subculture with HEPES Buffered Saline Solution, Trypsin/EDTA, and Trypsin Neutralizing Solution. Cells were maintained at 37 °C in a 5% CO2 atmosphere. All experiments were performed between passages 1–5.
Cc 4175
Manufactured by Lonza
The CC-4175 is a laboratory instrument designed for performing cell culture-related tasks. It functions as an incubator, providing a controlled environment for cell growth and maintenance.
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Lab products found in correlation
Cc 3171, by Lonza (3 mentions)
Cc 3170, by Lonza (2 mentions)
Cc 2540 lot 580580, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Bebm bronchial epithelial cell growth basal medium, by Lonza (1 mentions)
Begm bronchial epithelial cell growth medium, by Lonza (1 mentions)
Gentamicin amphotericin b, by Lonza (1 mentions)
Crl 2110, by American Type Culture Collection (1 mentions)
Cc 2540, by Lonza (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Corning (1 mentions)
Purecol type 1 bovine collagen solution, by Advanced BioMatrix (1 mentions)
Atcc crl 9609, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Begm singlequots, by Lonza (1 mentions)
Ccl 185, by American Type Culture Collection (1 mentions)
Htb 55, by American Type Culture Collection (1 mentions)
9 protocols using cc 4175
1
Culturing Normal Human Bronchial Epithelial Cells
2
Murine and Human Bronchial Cell Culture
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MLE-12 cells (CRL-2110, ATCC), a murine bronchial alveolar cell line, were cultured in complete DMEM. BEAS-2B cells (purchased from Procell Life Science & Technology Co. Ltd), a kind of human bronchial epithelium, were cultured in complete bronchial epithelial cell growth medium (Lonza, CC-3170 and CC-4175). The cells were incubated in a humidified atmosphere containing 95% O2 and 5% CO2 at 37°C. The cells were seeded in 24-well plates overnight and then stimulated with PBS or HDM at doses of 100 μg/mL for 24 hours.
3
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.
4
Culturing Human Bronchial Epithelial Cells
5
Culturing BEAS-2B Cells for Functional Assays
6
Nasal AEC Culture and Genetic Modification
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Nasal AECs were passaged twice using the modified Schlegel culture method17 (link), 18 (link). Prior to transduction, the nasal AECs were sub-cultured on collagen-coated 100mm dishes in BEGM growth media (CC-4175, Lonza) plus 10 μ M ROCK inhibitor for one to two days. Cells were then infected with either GFP, MUC18 CRISPR-Cas9, MUC18_Scr CRISPR-Cas9 lentiviral media (viral media diluted in BEGM media (total volume 8ml) plus 10 μ M ROCK inhibitor, 16 mM HEPES buffer, and 12 μg/ml polybrene) by spin infection (920 g at 25 degrees C) for 1 hour. At 24 hours post-infection puromycin (1 μg/ml final concentration) was added to the culture media. In parallel we transduced 3T3 fibroblasts with the empty plentyCRISPR vector and selected these cells (1 μg/ml puromycin) to generate feeder cells resistant to puromycin. The viral transduced nasal AECs were grown to 80-90% confluence and passaged using the modified Schlegel culture method and the puromycin resistant 3T3 cells. These modified Schlegel culture conditions were maintained throughout the selection period to generate transduced GFP expressing, MUC18 scrambled gRNA expressing, and MUC18 KO cells, as described in Figure 3 and Supplementary Figure 1 .
7
Culturing MDCK and NHBE Cell Lines
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MDCK and MDCK-NS1-GFP (Kochs et al., 2009 (link)) cells were maintained at 37°C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Gibco®) supplemented with 10% Fetal Bovine Serum (FBS, Corning®) as described elsewhere (Chua et al., 2013 (link)). Normal human bronchial epithelial (NHBE) cells (Lonza, CC-2540 Lot# 580580) were isolated from a 79 years-old Caucasian female and were maintained in bronchial epithelial growth media (Lonza, CC-3171) supplemented with BEGM SingleQuots as per the manufacturer’s instructions (Lonza, CC-4175) at 37°C and 5% CO2.
8
Nasal AEC Culture and Genetic Modification
9
Maintenance of Human Lung Cell Lines
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Human adenocarcinomic alveolar basal epithelial (A549) cells (ATCC, CCL-185), human adenocarcinomic lung epithelial (Calu-3) cells (ATCC, HTB-55), human HEp-2 cells (ATCC, CCL-23), Madin-Darby Canine Kidney (MDCK) cells (ATCC, CCL-34), MDCK-NS1 cells (García-Sastre et al., 1998 (link)) and African green monkey kidney epithelial Vero E6 cells (ATCC, CRL-1586) were maintained at 37°C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% Fetal Bovine Serum (FBS, Corning). Undifferentiated normal human bronchial epithelial (NHBE) cells (Lonza, CC-2540 Lot# 580580) were isolated from a 79-year-old Caucasian female and were maintained in bronchial epithelial growth media (Lonza, CC-3171) supplemented with BEGM SingleQuots as per the manufacturer’s instructions (Lonza, CC-4175) at 37°C and 5% CO2.
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