Beas2B (ATCC, Manassas, VA) were grown in BEGM (Lonza, Basel, Switzerland). A549 cells and A549 cells with Smad4 deletion (Sigma-Aldrich, St. Louis, MO) were grown in F12 supplemented with 10% FBS and 2mM L-glutamine. H1299 cells (ATCC) were grown in RPMI supplemented with 10% FBS. Cell lines with stable Smad4 knockdown were created by transfecting cells with pcPUR+U6 containing an anti-Smad4 shRNA (52 (link)) (kindly provided by Hideaki Ijichi, University of Tokyo, Japan) followed by puromycin selection; the pcPUR+U6 cassette (iGene Therapeutics, Tokyo, Japan) was used as a control. Reduced Smad4 expression was confirmed by RT-qPCR and western blot against Smad4 (1:200 #7966 Santa Cruz) with GAPDH control (1:40000, ab8245, Abcam, Cambridge, MA). Cell line identity was validated by DNA profiling at the University of Colorado DNA Sequencing & Analysis Core.
H1299 cells
Manufactured by American Type Culture Collection
Sourced in United States
H1299 cells are a human non-small cell lung cancer (NSCLC) cell line derived from the lymph node metastasis of a lung carcinoma. The cells are epithelial-like in morphology and are used in research applications to study lung cancer biology and evaluate potential therapeutic agents.
Automatically generated - may contain errors
Lab products found in correlation
A549 cell, by American Type Culture Collection (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Beas 2b, by American Type Culture Collection (3 mentions)
Bronchial epithelial growth medium (begm), by Lonza (2 mentions)
A549 cells with smad4 deletion, by Merck Group (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Rpmi media, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Hct116 cell, by American Type Culture Collection (2 mentions)
Hek293 cell, by American Type Culture Collection (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Solarbio (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
H1299 cells, by Thermo Fisher Scientific (2 mentions)
44 protocols using h1299 cells
1
Smad4 Knockdown in Cell Lines
2
Combination Therapy for Lung Cancer
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Doxorubicin, OSI-906, BYL719, binimetinib, and paclitaxel were purchased from LC Laboratories (Woburn, MA). PIM447 and LCL161 were obtained from Selleck Chemicals (Houston, TX). Vinorelbine was purchased from Sigma-Aldrich (St. Louis, MO). THZ1 was purchased from Cayman Chemical Company (Ann Arbor, MI). Human lung carcinoma PC9 cells were obtained from Sigma-Aldrich, and H1299 cells were provided from ATCC (Manassas, VA). All cell lines were grown in RMPI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, at 37 °C and 5% CO2.
3
Targeting HD-PTP to Modulate FAK Signaling
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SW1573 cells (ATCC) were grown in L-15(ATCC 30–2008) medium with 10% FBS, 2mM l-glutamine, and 1% penicillin-streptomycin. H1299 cells (ATCC) were grown in RPMI 1640 with 10% FBS, 2mM l-glutamine, and 1% penicillin-streptomycin. For transient knockdown, scrambled non-targeting (D-001210-01-05; siGENOME) and HD-PTP (PTPN23) siRNA (D-009417-01-0005; siGENOME; Horizon) were transfected using Lipofectamine RNAiMax reagent (Thermofisher). To generate stable HD-PTP knockdown cell lines we transduced SW1573 and H1299 cells with 3 individual HD-PTP shRNAs (LPP-HSH067569-LVRH1GH) and control lentiviral particles (Scramble, LPP-CSHCTR001-LVRH1GH) (GeneCopoeia). Cells were selected with hygromycin and the strongest HD-PTP shRNA knockdown was used for the remaining experiments. The FAK inhibitor defactinib (VS-6063) was purchased from Selleckchem.
4
Characterization of NSCLC Cell Lines
5
PF-573228 Cytotoxicity Assay
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A549 cells, H1299 cells, and H460 cells were purchased from ATCC. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere at 5% CO2, and treated with PF-573228 (TOCRIS, Bristol, UK) at concentrations of 0, 0.1, 1, or 10 μM.
6
Prostaglandin E2 Release Assay
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Beas2B and A549 cells (ATCC) were grown in DMEM supplemented with 5% fetal bovine serum and 4 mM glutamine at 37°C in a 5% CO2 incubator. H1373 and H1975 cells (ATCC) were grown in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% fetal bovine serum and 4 mM glutamine at 37°C in a 5% CO2 incubator. H1299 cells (ATCC) were grown in Minimal Essential medium (MEM) supplemented with 10% fetal bovine serum and 4 mM glutamine at 37°C in a 5% CO2 incubator. Please note that our strain of Beas2B cells does not cause tumors in nude mice (Jiang et al. 2008 (link)).
For prostaglandin E2 (PGE2) release, cells were incubated in serum-free media and stimulated with 4 μM calcium ionophore (A23817, Sigma-Aldrich) 30 min prior to supernatant collection (Gerristen et al. 1987 (link)). As a control for COX inhibition, cells were treated with 30 μM indomethacin (Sigma-Aldrich), a known COX-1 and COX-2 inhibitor, 2 h prior to calcium ionophore stimulation.
For prostaglandin E2 (PGE2) release, cells were incubated in serum-free media and stimulated with 4 μM calcium ionophore (A23817, Sigma-Aldrich) 30 min prior to supernatant collection (Gerristen et al. 1987 (link)). As a control for COX inhibition, cells were treated with 30 μM indomethacin (Sigma-Aldrich), a known COX-1 and COX-2 inhibitor, 2 h prior to calcium ionophore stimulation.
7
Transfection of Human Cancer Cell Lines
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H1299 cells (ATCC) were grown in DMEM + 10% FBS (Sigma or Gibco) and transfected by Lipofectamine 2000 (Invitrogen) - 3 μl of the reagent per 3 μg total plasmid DNA for a 12-well plate well at approx. 80% cell confluence (and proportionally for other vessel sizes, including Nunc 8-well LabTek chambers). H358 cells (ATCC) were grown in RPMI + 10% FBS (Gibco) and transfected 2x by Lipofectamine 2000 (Invitrogen) - 6 μl of the reagent per 5 μg total plasmid DNA for a 6-well plate well at approx. 80% cell confluence and for the second time 24h later. MEF cells (TP53 -/-, MDM2 -/-) were a kind gift of prof. U. Hibner (Institut de Génétique Moléculaire de Montpellier, France), were grown in DMEM + 10% FBS (Sigma) and were transfected by Effectene (Qiagen) according to the manufacturer's manual. MCF7, SkBr3 and MDA-MB-468 (ATCC) cells were grown in DMEM + 10% FBS (Sigma).
8
Smad4 Knockdown in Cell Lines
9
Optimized EV Collection from Cell Lines
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HEK293T cells (ATCC) were cultured at 37 °C and 5% CO2 in high glucose DMEM supplemented with 10% FBS and 2 mM L-glutamine (all Gibco). H1299 cells (ATCC) were cultured under the same conditions using RPMI media (Gibco) and HCT116 cells (ATCC) in McCoys 5 A media (Gibco). Expi293F™ cells (Thermo Fisher Scientific) were cultured in synthetic Expi293 expression media™ (Thermo Fisher Scientific) at 37 °C at 125 rpm in 2 litre Corning® roller bottles (Sigma).
For EV collections, adherent cell culture media was supplemented with 10% exosome depleted serum (Thermo Fisher Scientific) 48 hours prior to collection. For Expi293F™ cells no supplementation was necessary. For EV collection technique comparison experiments HEK293T cells were plated in Nunc™ triple layer flasks for 24 hours (Thermo Fisher Scientific), washed twice with PBS and supplemented with 70 ml DMEM with 10% exosome depleted serum. Conditioned media was harvested from 3 triple layer flasks (210 ml total volume), from an average of 6.4*108 cells, for each EV collection technique. In AIEX optimisation experiments, for optimal experimental speed, scalability and ease of cell culture handling, cells were plated into Corning® 10 layer CellSTACKs® (Sigma) into 1 litre of DMEM containing 10% exosome depleted serum. Cells conditioned media for 48 hours before EVs were harvested from an average of 7.8*108 cells.
For EV collections, adherent cell culture media was supplemented with 10% exosome depleted serum (Thermo Fisher Scientific) 48 hours prior to collection. For Expi293F™ cells no supplementation was necessary. For EV collection technique comparison experiments HEK293T cells were plated in Nunc™ triple layer flasks for 24 hours (Thermo Fisher Scientific), washed twice with PBS and supplemented with 70 ml DMEM with 10% exosome depleted serum. Conditioned media was harvested from 3 triple layer flasks (210 ml total volume), from an average of 6.4*108 cells, for each EV collection technique. In AIEX optimisation experiments, for optimal experimental speed, scalability and ease of cell culture handling, cells were plated into Corning® 10 layer CellSTACKs® (Sigma) into 1 litre of DMEM containing 10% exosome depleted serum. Cells conditioned media for 48 hours before EVs were harvested from an average of 7.8*108 cells.
10
Culturing Mouse Embryonic Stem Cells and Human Cell Lines
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E14 ESCs (female, 129/Ola) were obtained from the LMS Transgenics and ES cell facility. The ESCs were maintained on gelatin-coated plates, at 37°C, 5% CO2, in KnockOut DMEM supplemented with 15% fetal bovine serum (FBS), 1× NEAA, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, amd 100 μM β-mercaptoethanol (β-ME) (all reagents from Thermo Fisher Scientific, USA) and 1000 U/ml LIF (Merck, USA). HEK293 cells and A549 cells (American Type Culture Collection [ATCC]) were maintained in DMEM supplemented with 10% FBS, 2 mM l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. H1299 cells (ATCC, USA) were maintained in RPMI-1640 media supplemented with the same constituents as mentioned above.
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