Immortalized mouse hypothalamic cell line N39 (mHypoE-39) was obtained from CELLutions Biosystems and maintained in Dulbecco’s modified Eagle medium (DMEM) (Sigma) supplemented with 10 % fetal bovine serum (FBS) (Hyclone Laboratories) and 1 % penicillin/streptomycin (GIBCO). Lenti-X™ 293 T cell line was purchased from Clontech and cultured in DMEM supplemented with 10 % FBS and 1 % penicillin/streptomycin. Recombinant mouse Wnt3a (1324-WN, R&D Systems) was dissolved in phosphate-buffered saline (PBS) containing 0.2 % BSA. BIO (Sigma), a specific GSK3 inhibitor, was dissolved in dimethyl sulfoxide (DMSO) at 1 mM concentration.
Lenti x 293t cell line
Manufactured by Takara Bio
Sourced in United States, Japan
The Lenti-X 293T cell line is a human embryonic kidney cell line that is optimized for the production of lentiviral particles. This cell line provides efficient production of high-titer lentiviral stocks, making it a useful tool for lentiviral transduction and gene delivery applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Lenti x concentrator, by Takara Bio (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Polybrene, by Merck Group (4 mentions)
Pspax2, by Addgene (4 mentions)
Pmd2 g, by Addgene (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Sodium pyruvate, by Merck Group (3 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Lenti x qrt pcr titration kit, by Takara Bio (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Lenti x gostix, by Takara Bio (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
75 cm2 flask, by Corning (2 mentions)
86 protocols using lenti x 293t cell line
1
Wnt Signaling Pathway Activation in Mouse Hypothalamic Cells
2
Lentiviral Particle Production for Gene Delivery
3
Lentiviral Vector Production and Transduction
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The Lenti-X 293T cell line (Clontech) was maintained in high-glucose Dulbecco's Modified Eagle Medium (Gibco) supplemented with 100 U/ml penicillin (Sigma-Aldrich), 100 mg/ml streptomycin (Sigma-Aldrich), 0.5% sodium pyruvate (Sigma-Aldrich), and 10% FBS (Sigma-Aldrich). Lenti X cells were cultured in 75 cm2 flasks (Corning) and passed every 2–3 days. Lentiviral vectors were produced using a 3rd generation lentiviral system consisting of pMDLg/pRRE, pRSV-Rev, pMD2.G (the plasmids were a gift from Didier Trono (Addgene plasmid # 12251, 12253, 12259)), and pLV/mIL-12, plasmid encoding murine il12 gene (p35 and p40 subunits); pLV/mIL-18, plasmid encoding murine il18 gene; pLV/mIL-18/mIL-12, plasmid encoding murine il-18 and il12 gene (p35 and p40 subunits) simultaneously; and pLV/EGFP, control plasmid encoding enhanced green fluorescent protein (EGFP). Lentiviral vectors used for cell modifications were produced as previously described by Rossowska et al. [26 (link)]. LV-containing supernatant was collected and concentrated by PEG 6000 (Sigma-Aldrich) precipitation. The pellet containing lentiviral vectors was suspended in PBS and stored at -80°C. The titer of the lentiviral vectors was evaluated using the commercially available QuickTiter Lentivirus Titer Kit according to the manufacturer's instructions (Cell Biolabs).
4
Induced Pluripotent Stem Cell Generation
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Lenti-X™ 293T cell line (Clontech, Cat# 632180) was maintained in DMEM medium plus10% FBS and 1% Pen Strep. Mouse embryonic fibroblasts (MEFs) from various mice strains were prepared from E13.5 embryos and maintained in DMEM medium plus 10% FBS and 1% Pen Strep. MEFs used for reprogramming were within passage three. For reprogramming, MEFs were cultured in reprogramming medium (DMEM with 15% FBS, 1% Pen Strep, Glutamax, Sodium Pyruvate, MEM Non-Essential Amino Acids, 0.1mM β-mercaptoethanol (Thermo Fisher Scientific), 1000 U ml−1 LIF (Millipore Sigma, Cat# ESG1106), and 2μg ml doxycycline). Mouse embryonic stem cells (ESCs) were prepared from mouse blastocysts at embryonic day 3.5 (E3.5)45 (link). Mouse ESCs and iPSCs were cultured in reprogramming medium and grown on MEF feeder cells. For feeder-free culture, mouse ESCs and iPSCs were cultured in Knockout DMEM medium plus 20% KOSR (Thermo Fisher Scientific, Cat# 10828028), 1% Pen Strep, Glutamax, Sodium Pyruvate, MEM Non-Essential Amino Acids 0.1mM, β-mercaptoethanol, 1000 U ml−1, 3 μM CHIR99021 (Selleckchem, Cat# S1263) and 1μM of PD0325901 (Selleckchem, Cat# S1036). To induce MEFs into neurons, MEFs within three passages were infected with Ascl1 lentivirus construct and cultured in N3 medium for neuronal differentiation33 (link). Cells were free of mycoplasma.
5
Culturing Human Cancer Cell Lines
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Human cancer cell lines (HCT-116, HT-29) were purchased from the American Type Culture Collection. Lenti-X 293T cell line was purchased from Clontech Laboratories (Palo Alto, CA, USA). Cell lines were cultured in DMEM with added fetal bovine serum, glutamine, penicillin, and streptomycin. The absence of mycoplasma contamination was confirmed using Myco Alert (Lonza, Basel, Switzerland).
6
Cultivation of MDCK and HEK293T Cell Lines
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Madin-Darby Canine Kidney (MDCK-ATL) Cells, FR-926, and Madin-Darby Canine Kidney (MDCK) Cells, London Line, FR-58, were obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA. The human embryonic kidney 293T cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA), and the Lenti-X 293T cell line was obtained from Clontech (Mountain View, CA). Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Lonza, Allendale, NJ) containing 10% fetal bovine serum (FBS; Thermo Scientific, Waltham, MA), 100 IU/ml penicillin, and 100 µg/ml streptomycin. All cells were incubated at 37°C and 5% CO2. Influenza virus-infected cells were grown in DMEM containing 0.5% bovine albumin and TPCK trypsin (Sigma-Aldrich, St. Louis, MO).
7
Lentiviral Reporter for GAS Signaling
8
Lentiviral Knockdown of β-Catenin
9
Ubiquitin Regulation in Neurodegeneration
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15dPGJ2 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Antibody sources were as follows: mouse monoclonal anti-mono- and poly-ubiquitinated proteins antibody (clone FK2) and anti-poly-ubiquitinated proteins antibody (clone FK1) were from Enzo Life Sciences (Plymouth Meeting, PA, USA). Monoclonal anti-ubiquitin antibody was from Covance (Berkeley, CA, USA) and anti-ubiquitin Lys48-specific antibody was from Millipore (Temecula, CA, USA), anti-ubiquitin Lys63-specific antibody was from Abcam (Cambridge, MA, USA); anti-PARP, anti-caspase-3, and anti-Neurofilament-L antibodies were from Cell Signaling (Boston, MA, USA); anti-β-actin antibody and anti-UCH-L1 antibody were from Sigma-Aldrich (St Louis, MO, USA); anti-NeuN antibody was from Millipore; and anti-GAPDH antibody was from Ambion (Grand Island, NY, USA). Cy3- and Alexafluor 488-conjugated secondary antibodies were from Jackson Immunoresearch Lab (West Grove, PA, USA). Ultra performance liquid chromatography organic solvents and water were from VWR (West Chester, PA, USA). The lentiviral expression vector, pLVX-IRES-ZsGreen1 vector, and Lenti-X HTX concentrator and packaging system were purchased from Clontech Laboratories (Mountain View, CA, USA). WST-1 cell proliferation assay kit and Lenti-X 293 T cell line were purchased from Clontech.
10
Proteomic analysis of INF2-associated proteins in human podocytes
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GFP–INF2–WT was subcloned into pWPXL [pWPXL was a gift from Didier Trono (École polytechnique fédérale de Lausanne, Addgene plasmid # 12257)] and together with packaging vectors pMDG.2 and psAX2 [pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259) and psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260)] transfected into Lenti-X 293T Cell Line (Clontech). Human podocytes were transduced with GFP or GFP–INF2 lentivirus with 8 μg/ml polybrene overnight, the cells were thermo switched and differentiated for a minimum of 10 days [18 (link)]. 2× 175 tissue culture flasks were lysed and GFP and GFP–INF2 protein and interacting proteins were immunoprecipitated using the GFP-Trap system (Chromotek). Samples were separated on Nupage 4–12% precast gels (Invitrogen) and subjected to LC–MS/MS analysis on an Orbitrap Velos (Thermo) mass spectrometer as described previously [19 (link),20 (link)]
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