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Nci h441 cells

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NCI-H441 cells are a human lung adenocarcinoma cell line derived from a 65-year-old Caucasian male. These cells express markers characteristic of Clara cells and have the ability to differentiate into type I and type II alveolar cells. They can be used for research in lung cancer, respiratory biology, and toxicology.

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8 protocols using nci h441 cells

1

Cell Culture Conditions for NCI-H441 and A549-hACE2

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The human lung adenocarcinoma NCI-H441 cells were purchased from ATCC and maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA, USA), 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin. The A549-hACE2 (HA-Flag) cells were obtained from BEI Resources (NIAID, NIH) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Atlanta Biologicals), 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin and 1 μg/mL puromycin. The cells were grown at 37 °C and 5% CO2 in a humidified chamber.
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2

Endothelial and Fibroblast Cell Isolation

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The use of embryos for cell culture was approved by the University of Queensland animal ethics committee (SCMB/002/18). Seventeen-day-old chicken and twenty-one-day-old Pekin duck embryonated eggs were purchased from Darling Downs Hatchery (Queensland, Australia). Primary aortic endothelial cells were cultured from the aortic arches of chicken and duck embryos as described previously using EGM-2MV medium (Lonza, Basel, Switzerland) with 10% FCS (Gibco, Waltham, MA, USA) [7 (link),8 (link)]. Primary chicken and duck embryo fibroblasts were cultured as described previously [9 (link),10 (link)]. Madin–Darby Canine Kidney (MDCK) cells were obtained from American Type Culture Collection (ATCC), and cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% FCS (Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (Gibco, Waltham, MA, USA). NCI-H441 cells (human lung epithelial cells) were obtained from ATCC and cultured in RPMI (Gibco, Waltham, MA, USA) medium supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco). Primary human pulmonary microvascular endothelial cells were obtained from Sciencell (Carlsbad, CA, USA) and cultured in an endothelial cell growth medium (Sciencell, Carlsbad, CA, USA). All cell lines of mammalian origin were incubated at 37 °C 5% CO2 whilst all avian cells were cultured at 40 °C 5% CO2 unless otherwise stated.
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3

In Vitro PMN Transmigration Assay

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We performed the in vitro transmigration assay of human PMNs through a monolayer of NCI-H441 cells (ATCC, USA) (n≥2) to separate the effects of the PDE4-inhibitors on PMNs and epithelium [23 (link)]. Epithelium or PMNs were incubated with rolipram or roflumilast for 60 min at indicated concentrations (n≥4). Until reaching confluence, human epithelial cells were cultivated on inserts of a transwell system (3.0μm pore size, 6.5mm diameter; Costar, Cambridge, MA, USA). Isolated human PMNs (Percoll gradient; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) migrated through the monolayer of epithelial cells along a chemotactic gradient (CXCL2/3; 200ng/ml; Pepro Tech, Hamburg, Germany). By determination of myeloperoxidase were migrated PMNs quantified (absorption length: 405 nm).
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4

Cell Culture Conditions for Various Cancer Cell Lines

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EBC-1 cells (JCRB) were cultured in MEM Eagle medium (Sigma-Aldrich) with 10% FBS (Invitrogen) and 2 mM L-glutamine (Invitrogen). A549 (ATCC), MDA-MB-453 (provided by Danny R. Welch from the Jake Gittlen Cancer Research Institute) and MDA-MB-231 cells (ATCC) were cultured in DMEM (Gibco, Invitrogen) with 10% FBS. NCI-H441 cells (ATCC) were cultured in RPMI 1640 medium (Invitrogen) with 2.5 g/L D(+)-glucose (Sigma-Aldrich), 10 mM HEPES (Gibco, Invitrogen), 2 mM L-glutamine, 1 mM sodium pyruvate (Gibco, Invitrogen) and 10% FBS. Jurkat E6.1 cells were cultured in RPMI 1640 medium with 2 mM glutamine, 1 mM sodium pyruvate and 10% FBS. Cell lines were cultured at 37°C and 5% CO2. Only MDA-MB-231 cells were cultured at 10% CO2. The MDA-MB-435 cell line was originally reported as breast cancer cell line. Recent studies provided evidence for MDA-MB-435 being a melanoma cell line [46 (link)]. In this study, MDA-MB-435 cells were used as negative control for c-Met binding. Data interpretation in this study is therefore not affected by the reported misidentification of this cell line.
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5

Cell Culture Conditions for Caco2, NCI-H441, and SHSY5Y Cell Lines

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Caco2 cells (ATCC, Manassas, VA, USA) were grown in DMEM with 4.5 g/L glucose, l-glutamate and sodium pyruvate supplemented with 10% Fetal Bovine Serum (FBS, Corning, Cat# 35-010-CV), 1% penicillin and streptomycin, 25 µg/mL amphotericin B, and 2 mM L-Glutamine. NCI-H441 cells (ATCC) were grown in RPMI-1640 (Corning, Cat# 10-040-CV) with l-glutamine, supplemented with 10% FBS, 1% penicillin and streptomycin (EuroClone, Cat# ECB3001D), 25 µg/mL amphotericin B (Corning, Cat# 30-003-CF), and 2 mM l-glutamine (EuroClone, Cat# ECB3000D). SHSY5Y cells (kindly provided by Dr. Maria Lasalvia, University of Foggia) were grown in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 Ham (Sigma-Aldrich, Cat# D8062), 20% FBS, 1% non-essential amino acids, 2 mM l-glutamine, 1% penicillin and streptomycin, and 25 µg/mL amphotericin B. The cells were incubated at 37 °C in a humidified incubator with 5% CO2 and the culture medium was refreshed every other day.
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6

Cell Culture Protocols for NCI-H441, HPMEC, and MDCK

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NCI-H441 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in RPMI medium (Gibco, Grand Island, NE) with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). NCI-H441 cells were used between passages 2 and 14. Primary human pulmonary microvascular endothelial cells (HPMECs) were obtained from Sciencell (Carlsbad, CA) and cultured in endothelial cell growth medium (Sciencell). HPMECs were used at passages 2 to 7. Madin-Darby canine kidney (MDCK) cells were obtained from ATCC and kept in Dulbecco modified Eagle medium (DMEM; Gibco) with 10% FBS and 1% penicillin-streptomycin. MDCK cells were used between passages 20 and 50. All cell lines were cultured using a humidified 37°C incubator with 95% O2 and 5% CO2.
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7

NCI-H441 Cell Culture with Methylprednisolone

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NCI-H441 cells (American Type Culture Collection, Manassas, Va) were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin (Invitrogen, Carlsbad, Calif). Cells were grown in T-25 flasks at 37 C in 5% CO 2 humidified atmosphere. Cells during passages 10 to 15 were plated at approximately 80% confluence and cultured for 24 hours using incremental doses (10 À5, À6, À7, À8, À9 M) of methylprednisolone (Sigma, St Louis, Mo). Control cells received the vehicle 0.01% (v/v) dimethyl sulfoxide.
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8

Cell Line Cultivation Protocols

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NCI-H441 cells (lot no. 58294188), a human lung epithelial cell line with characteristics of Clara cells, and RLE-6TN cells (lot no. 59111690), a rat lung epithelial cell line with characteristics of alveolar type II cells, were purchased from the American Type Culture Collection (Rockville, MD, USA) in 2010 and 2013, respectively. All experiments using these cells were performed within 4 months after resuscitation. NCI-H441 cells were grown as described in our previous report [4 (link)]. RLE-6TN cells were grown in Ham’s F12 medium containing 2 mM L-glutamine (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum, 10 μg/ml bovine pituitary extract (PromoCell, Heidelberg, Germany), 5 μg/ml insulin (Gibco), 2.5 ng/ml insulin-like growth factor (Sigma-Aldrich, St. Louis, MO, USA), 1.25 μg/ml transferrin (Gibco), and 2.5 ng/ml epidermal growth factor (Sigma-Aldrich, St. Louis, MO, USA). COS7 and NIH3T3 cells were described previously [14 (link), 15 (link)]. COS7 cells that overexpressed CADM1 exogenously were described previously [11 (link)].
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