The human pulmonary alveolar epithelial cell line NCI-H441 was obtained from ATCC (Manassas, VA, USA), and cultured in RPMI 1640 medium containing 2.5 g/L dextrose, 2.383 g/L HEPES, 0.11 g/L sodium pyruvate, 10% FBS (v/v), 2.2 g/L sodium bicarbonate, 100 units/mL of penicillin and streptomycin. Cells were plated at 2.4 × 105 cells/mL in RPMI 1640 medium. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
Nci h441
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom
NCI-H441 is a human lung adenocarcinoma cell line derived from a 65-year-old Caucasian male. It is commonly used in research for studying lung cancer and related biological processes. The cell line exhibits epithelial-like morphology and expresses markers typical of lung adenocarcinoma.
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Lab products found in correlation
Nci h1975, by American Type Culture Collection (17 mentions)
Nci h1299, by American Type Culture Collection (14 mentions)
Nci h358, by American Type Culture Collection (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Nci h460, by American Type Culture Collection (11 mentions)
Nci h23, by American Type Culture Collection (11 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Nci h2228, by American Type Culture Collection (6 mentions)
Nci h1650, by American Type Culture Collection (6 mentions)
Calu 3, by American Type Culture Collection (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Nci h520, by American Type Culture Collection (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Mda mb 231, by American Type Culture Collection (5 mentions)
Beas 2b, by American Type Culture Collection (5 mentions)
Hcc827, by American Type Culture Collection (5 mentions)
Nci h2347, by American Type Culture Collection (4 mentions)
Nci h1734, by American Type Culture Collection (4 mentions)
Calu 1, by American Type Culture Collection (4 mentions)
60 protocols using nci h441
1
NCI-H441 Alveolar Epithelial Cell Culture
2
Cell Line Authentication and Validation
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The NCI‐H23, NCI‐H441, NCI‐H1299, and NCI‐H2009 lung adenocarcinoma cell lines were purchased from ATCC (Manassas, VA, USA), whereas PC‐9 was obtained from RIKEN Cell Bank (Tsukuba, Japan). ACC‐LC‐319 and ACC‐LC‐94 lung adenocarcinoma cell lines were established by our group. An immortalized lung epithelial cell line, BEAS‐2B, was a generous gift from Curtis C. Harris (National Cancer Institute, Bethesda, MD, USA). The conditions used to culture these cell lines have been previously reported.18 Verification of all cell lines was carried out by short tandem repeat profiling at the Japanese Collection of Research Bioresources, National Institute of Biomedical Innovation of Japan (Osaka, Japan) in February 2015. All cell lines were confirmed to be absent of mycoplasma contamination (MycoAlert; Lonza, Tokyo, Japan).
3
Culturing Human Lung Cancer Cell Lines
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Four human lung cancer cell lines were used in this study. PC9 (Riken BioResource Research Center), NCI‐H441 (ATCC), and NCI‐H1975 (ATCC) were cultured in RPMI‐1640 medium (Wako Pure Chemical Industries) supplemented with 10% FBS (Thermo Fisher Scientific) at 37°C and 5% CO2. A549 (ATCC) was cultured in EMEM (Wako Pure Chemical Industries) supplemented with 10% FBS and 1% nonessential amino acids (Wako Pure Chemical Industries) at 37°C and 5% CO2.
4
In Vitro Transmigration Assay of Human PMNs
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We performed the in vitro transmigration assay of human PMNs through a monolayer of pulmonary epithelial (NCI-H441, ATCC, USA) (n≥3) or primary pulmonary endothelial cells (HMVEC-L, Lonza Walkersville, Walkersville, MD, USA). Endothelial (respectively epithelial) cells, PMNs, or both were incubated with the specific CXCR4 or CXCR7 antagonist at indicated concentrations. Human endothelial/epithelial cells were cultivated on inserts of a transwell system (3.0-μm pore size, 6.5-mm diameter; Costar, Cambridge, MA, USA) until reaching confluence. Isolated human PMNs (Percoll gradient; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) migrated through the monolayer of endothelial/epithelial cells along a chemotactic gradient (CXCL2/3; 200 ng/ml; Pepro Tech, Hamburg, Germany). Migrated PMNs were quantified by determination of myeloperoxidase (absorption length: 405 nm) in the bottom wells. Additionally, migrated PMNs were evaluated by modified Giemsa staining (Diff Quik) by two experienced independent observers.
5
Cell Culture of Diverse Cell Lines
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ISO‐HAS‐1 (human microvascular endothelial cell line, originated from (Masuzawa et al., 1999 ; Unger et al., 2002 )), NCI H441 (human lung adenocarcinoma cell line), A549 (human lung carcinoma cell line) and THP‐1 (human leukaemia monocyte cell line; all three cell lines purchased from ATCC, ATCC‐HTB‐174, CCL‐185 and TIB‐202, Promochem, Wesel, Germany) were grown in RPMI 1640 (Gibco) supplemented with l ‐glutamine, 10% fetal calf serum (FCS) and penicillin–streptomycin (Pen/Strep; 100 U/100 µg/ml) and cultivated at 37 °C in 5% CO2. ISO‐HAS‐1 and H441 were passaged every third day at a dilution of 1:3 until passages 50 and 35, respectively, and THP‐1 were passaged with a cell number of 1 × 106–1 × 105 cells/ml.
6
Proteasomal and Lysosomal Inhibitors in Cell Lines
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NCI‐H1975, NCI‐H441, NCI‐H1299, NCI‐H2228, HCC4006, and HeLa cells were purchased from ATCC, and PC‐9 cells were obtained from Riken Cell Bank. All cell lines were cultured in RPMI‐1640 medium with 10% FBS. Cell lines were authenticated by short tandem repeat DNA profiling and were free from mycoplasma contamination.
For treatment with the proteasomal inhibitor MG132 and the lysosomal inhibitor chloroquine, a total of 1 × 105 cells per well were seeded in 6‐well plates and treated 24 hours later with GA, MG132, chloroquine, or DMSO at the indicated concentrations. The cells were harvested 8 hours after treatment for western blot analysis.
For treatment with the proteasomal inhibitor MG132 and the lysosomal inhibitor chloroquine, a total of 1 × 105 cells per well were seeded in 6‐well plates and treated 24 hours later with GA, MG132, chloroquine, or DMSO at the indicated concentrations. The cells were harvested 8 hours after treatment for western blot analysis.
7
Comprehensive Cell Line Analysis
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All cell lines were obtained from the Genentech cell bank, gCELL. The cell lines used for this study included were MOR (ECACC), NCI-H2122 (ATCC), A549 (ATCC), NCI-H441 (ATCC). Small Molecule inhibitors were either purchased from outside vendors or generated at Genentech. S1 Table lists the inhibitor name, expected target, source and relevant catalogue number.
8
Cell Line Maintenance and Modifications
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The A549, Calu-6, Colo205, DU-145, HCC1569, HEK293, JIMT-1, MDA-MB-231, MDA-MB-453, NCI-H441, NCI-H1569, NS-1, PC-3, SK-BR-3, SP2/0 and 22RV-1 cell lines used were purchased from ATCC and maintained in the recommended medium. HEK293-ERBB3 cells were purchased from Kyinno Biotechnology (KC-1496). SP2/0-HER3 cells stably expressing human HER3 were generated in house by transduction of human HER3 with lentivirus construct.
9
NSCLC Tumor Characterization Protocol
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40 patients with pathologically confirmed NSCLC were enrolled for the study from The Affiliated Hospital of Qingdao University from Jan, 2015 to Feb, 2017. The informed written consent had been signed by all patients and the hospital had approved the study. Tumors were histologically graded according to the American Joint Committee on Cancer (AJCC, version 8) [10 (link)]. The human lung epithelial cell BEAS-2B, and lung cancer cell lines NCI-H441, PC-9, NCI-H1650, A549 were purchased from ATCC. All cell lines were cultured in DMEM supplemented with 10% FBS at 37°C under 5% CO2.
10
Culturing Various Cancer Cell Lines
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Breast cancer cell line MDA-MB-231 cells, pancreatic cancer cell line PANC-1, lung adenocarcinoma cell lines NCI-H441, NCI-H661, NCI-H2228, colon adenocarcinoma cell line HT-29, non-small cell lung carcinoma cell line NCI-H1975, glioblastoma cell line U87, and acute lymphoblastic leukemia cell line CCL-119 were purchased from ATCC. All cells passed the mycoplasma contamination test. MDA-MB-231, PANC-1, NCI-H441, NCI-H1975, and U87 were cultured in DMEM (Corning, USA). HT-29 cells were grown in McCoy’s 5a Modified Medium (Gibco, USA). NCI-H661, NCI-H2228, and CCL-119 cells were maintained in RPMI 1640 medium containing 25 mM HEPES and L-glutamine (GE Healthcare, USA). All mediums were supplemented with 5% (v/v) EV-depleted Fetal Bovine Serum (FBS) (Thermal Fisher, USA), 100 units/ml penicillin, 100 μg/ml streptomycin, and 1% non-essential amino acid. All cell lines were incubated at 37 °C with 5% CO2 and a 95% humidified atmosphere.
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