Nsclc cell line

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NSCLC cell lines are a collection of human lung cancer cell lines derived from non-small cell lung carcinoma (NSCLC) patients. These cell lines serve as valuable research tools for studying the biology and treatment of this type of lung cancer.

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Humam NSCLC cell-lines A549 Human NSCLC cell lines H1299 NSCLC cell lines A549 NSCLC cell line A549 human NSCLC cell line

9 protocols using nsclc cell line

NSCLC Cell Line and Patient Samples

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NSCLC cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS.
Human specimens were obtained during surgery under a protocol approved by the Daping Hospital and Research Institute of Surgery review boards. NSCLC patients were divided into miR-150-5p high and low groups, as described previously.³⁵ (link) Briefly, mature miR-150-5p and the RNU6 endogenous control were analyzed using the TaqMan microRNA Assay Kit. The relative expression of miR-150-5p was normalized against RNU6 expression using the 2^_ΔCt method, and the miR-150-5p expression fold change in lung cancer samples matched to nontumor control samples was evaluated using the 2^_ΔCt method. On the basis of the mean fold change of miR-150-5p expression, patients were divided into miR-150-5p high- (fold change > mean) and low- (fold change < mean) expression groups. The demographic information of the cohort is presented in Table 1.

Dai F.Q., Li C.R., Fan X.Q., Tan L., Wang R.T, & Jin H. (2019). miR-150-5p Inhibits Non-Small-Cell Lung Cancer Metastasis and Recurrence by Targeting HMGA2 and β-Catenin Signaling. Molecular Therapy. Nucleic Acids, 16, 675-685.

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NSCLC Cell Culture Protocol

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NSCLC cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 medium from Gibco/BRL supplemented with 10% (v/v) fetal bovine serum at 37°C with 5% CO2.

Kanteti R., El-Hashani E., Dhanasingh I., Tretiakova M., Husain A.N., Sharma S., Sharma J., Vokes E.E, & Salgia R. (2014). Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer. BMC Cancer, 14, 185.

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Culturing of NSCLC Cell Lines

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The H226 cells were cultured in 1640 medium supplemented with 10% bovine pituitary extract. The H520 cells, A549 cells and PC9 cells were cultured in DMEM medium supplemented with 10% bovine pituitary extract. NSCLC cell lines were obtained from the American Type Culture Collection (ATCC) and were tested and authenticated by DNA typing at Shanghai Jiao Tong University Analysis Core. These cells were all cultured at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO₂.

Guo W., Kuang Y., Wu J., Wen D., Zhou A., Liao Y., Song H., Xu D., Wang T., Jing B., Li K., Hu M., Ling J., Wang Q, & Wu W. (2020). Hexokinase 2 Depletion Confers Sensitization to Metformin and Inhibits Glycolysis in Lung Squamous Cell Carcinoma. Frontiers in Oncology, 10, 52.

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NSCLC Cell Culture Optimization

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NSCLC cell lines were obtained from American Type Culture Collection (ATCC, LGC Standards, Wesel, Germany). A549 cells were cultured in high glucose F-12 K (Kaighn’s Modification of Ham’s F-12 Medium) mixture (GIBCO-BRL Co, NY, USA), supplemented with 10% fetal bovine serum (FBS) (GIBCO-BRL) and 1% penicillin/streptomycin (P/S) (GIBCO-BRL). The H1975 cell culture medium was D-MEM 4.5 g/L D-glucose (GIBCO-BRL) with 10% FBS/ 1% P/S. SKMES-1 cells were cultured in MEMα (GIBCO-BRL) medium supplemented with 10% FBS/1% P/S. Cells were maintained in a humidified atmosphere of 5% CO₂⁻ 95% air at 37 °C, and sub-cultivation was performed using ethylenediaminetetraacetic acid (EDTA)/Trypsin 0.25% (GIBCO-BRL).
Cytospins of H1975 cells and interferon gamma (IFN-γ)-treated A549 cells were also prepared to serve as controls for the optimization of the immunofluorescence stainings.

Papadaki M.A., Sotiriou A.I., Vasilopoulou C., Filika M., Aggouraki D., Tsoulfas P.G., Apostolopoulou C.A., Rounis K., Mavroudis D, & Agelaki S. (2020). Optimization of the Enrichment of Circulating Tumor Cells for Downstream Phenotypic Analysis in Patients with Non-Small Cell Lung Cancer Treated with Anti-PD-1 Immunotherapy. Cancers, 12(6), 1556.

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Lung Cancer Samples Acquisition and Cell Line Development

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The Institute’s Ethics Committees approved this study and all samples were obtained with written informed consent from participating patients. Lung tumor-normal pairs from 50 NSCLC patients were obtained from frozen tumor banks at The University of New Mexico (UNM) and the Mayo Clinic. NBEC collected through diagnostic bronchoscopy and PBMC were obtained from cancer-free smokers (19 (link)). NSCLC cell lines were obtained from and authenticated by the American Type Culture Collection (Manassas, VA). Three immortalized HBEC lines (HBEC1, HBEC2, and HBEC14) were obtained from Drs. Shay and Minna, Southwestern Medical Center, Dallas, TX (20 (link)). Cell lines were maintained for a maximum of 6 months (24 passages). HBECs exposed to the tobacco carcinogens MNU and BPDE for 4–12 weeks and MB transformed HBECs are selected for growth in soft-agar following 12 weeks of MB exposure are (summarized in Supplemental Table 1) were previously derived (13 (link)).

Tellez C.S., Juri D.E., Do K., Picchi M.A., Wang T., Liu G., Spira A, & Belinsky S.A. (2016). MiR-196b is epigenetically silenced during the pre-malignant stage of lung carcinogenesis. Cancer research, 76(16), 4741-4751.

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Cell Culture for NSCLC Research

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MRC-5, Calu-1, Calu-3, and MCF-7 cells were cultured under standard conditions in DMEM, while A549, H460, ER3, and HCC827 cells were grown in RPMI-1640 medium, all supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL penicillin/streptomycin. All media and supplements were from Sigma-Aldrich (Milan, MI, Italy). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. All NSCLC cell lines were purchased from American Type Culture Collection (ATCC). A549, MCF-7, and HCC827-ER3 cells were kindly provided by Dr. Balazs Halmos (Columbia University Medical Center, New York, NY, USA).

Russo V., Paciocco A., Affinito A., Roscigno G., Fiore D., Palma F., Galasso M., Volinia S., Fiorelli A., Esposito C.L., Nuzzo S., Inghirami G., de Franciscis V, & Condorelli G. (2018). Aptamer-miR-34c Conjugate Affects Cell Proliferation of Non-Small-Cell Lung Cancer Cells. Molecular Therapy. Nucleic Acids, 13, 334-346.

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NSCLC and HNSCC Cell Line Cultivation

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All NSCLC cell lines were from American Type Culture Collection (ATCC) (Manassas, VA, USA). NSCLC cell lines A549, H1993, SK-LU-1, H226, H1703, H2170, SK-MES-1, SW900 and the nonmalignant and immortalized control cell line BEAS-2B, were cultured in RPMI 1640 medium (Gibco/BRL) supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine and 1% penicillin-streptomycin. HEK293 cells were cultured in DMEM medium (Gibco/BRL) supplemented with 10% (v/v) fetal bovine serum (FBS), L-glutamine and 1% penicillinstreptomycin. All HNSCC cell lines were obtained from indicated sources (supplementary table 1) All cell lines were cultured at 37C with 5% CO2. ALW-II-41-27 was purchased from MedChemExpress (Monmouth Junction, NJ 08852, USA). EphrinA1, soluble EPHA2 and SLIT2 were purchased from R&D Systems (Minneapolis, MN 55413, USA). EGF ligand was purchased from Stem cell technology.

Pang K.M., Srivastava S., Iida M., Nelson M., Liu J., Nam A., Wang J., Mambetsariev I., Mohanty A., McDaniel N.K., Behal A., Kulkarni P., Wheeler D.L., & Salgia R. (2020). The Ephrin receptor A2 and Roundabout Guidance Receptor 1 heterodimer: A potential theranostic for squamous cell carcinomas.

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HCC827 NSCLC Cell Line Culture

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The HCC827 human non-small cell lung cancer (NSCLC) cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with heat-inactivated 10% fetal bovine serum (HyClone; GE Healthcare, Chicago, IL, USA), 1% 10,000 U/ml penicillin, and 10,000 µg/ml streptomycin in a 37°C, 5% CO₂ humidified incubator. HCC827 control or Claudin-7 KD cell lines were previously established (10 (link)).

Kim D.H., Lu Q, & Chen Y.H. (2019). Claudin-7 modulates cell-matrix adhesion that controls cell migration, invasion and attachment of human HCC827 lung cancer cells. Oncology Letters, 17(3), 2890-2896.

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Murine Tumor Imaging with ICG

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Twenty female C57BL/6 mice were purchased from Charles River Laboratories and Jackson Laboratories. All mice were maintained in pathogen-free conditions and used for experiments at ages eight weeks or older. The Animal Care and Use Committees of the Children’s Hospital of Philadelphia, The Wistar Institute, and the University of Pennsylvania approved all protocols in compliance with the Guide for the Care and Use of Laboratory Animals. The metastatic murine non-small-cell carcinoma (NSCLC) cell line and the murine Lewis Lung Carcinoma (LLC) cell line were obtained from American Type Culture Collection (Manassas, VA) and cultured in high-glucose DMEM (Dulbecco’s Modified Eagle’s Medium, Mediatech, Washington, DC) supplemented with 10% fetal bovine serum (FBS; Georgia Biotechnology, Atlanta, GA), 1% penicillin/streptomycin, and 1% glutamine. Cell lines were regularly tested and maintained negative for Mycoplasma spp.Twenty mice were injected with 2 × 10⁶ LLC cells in their right flanks. After two weeks of growth, each mouse was injected with ICG via tail vein, as previously described and then imaged with the SPIIF (12 (link)).

Okusanya O.T., Madajewski B., Segal E., Judy B.F., Venegas O.G., Judy R.P., Quatromoni J.G., Wang M.D., Nie S, & Singhal S. (2014). Small Portable Interchangeable Imager of Fluorescence for Fluorescence Guided Surgery and Research. Technology in cancer research & treatment, 14(2), 213-220.

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