Hek293t

Manufactured by InvivoGen

Sourced in United States

HEK293T is a commonly used human embryonic kidney cell line that is highly transfectable. It serves as a versatile and widely recognized model system for various cellular and molecular biology applications.

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8 protocols using hek293t

Cell Line Cultivation Protocol

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The following cell lines were used in this study: human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs; male)^{53 (link)}, HeLa (American Type Culture Collection (ATCC) CCL-2), HEK293T (ATCC CRL-11268), T-REx-293 (Life Technologies), BS-C-1 (ATCC CCL-26) and RK13 (ATCC CCL-37). All cell lines were cultured in DMEM (Gibco), supplemented with 10% FBS (PAN Biotech) and 50 μg ml⁻¹ penicillin–streptomycin (Gibco). T-REx-293-derived cells that were stably transfected with pcDNA4/TO plasmids were further supplemented with 10 μg ml⁻¹ blasticidin (Thermo Fisher) and 100 μg ml⁻¹ zeocin (Gibco). HEK293T cells that were transduced to overexpress Myc-tagged proteins^{10 (link)} were supplemented with 1 μg ml⁻¹ puromycin (Invivogen). A complete list of cell lines is described in Supplementary Table 1.

Zhao Y., Lu Y., Richardson S., Sreekumar M., Albarnaz J.D, & Smith G.L. (2023). TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6. Nature, 620(7975), 873-880.

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Cell Culture Conditions for Virus Research

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A549 (Human lung epithelial cells, ATCC CCL-185, Manassas, VA), BHK-21 (Newborn Hamster kidney fibroblast cells, ATCC CCL-10) and HEK-293T (Human epithelial kidney cells, ATCC CRL-11268) were cultured in Dulbecco’s Modified Eagle Medium (D MEM) (11965–118, Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Thermo Scientific), 50 U/mL penicillin-streptomycin (15140–163, Gibco), plus 10% heat-inactivated FBS (Lonza). BHK-21 were also supplemented with 20% Tryptose Phosphate Broth (18050039, Thermo Scientific). HEK-293T cells were supplemented with sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM). HEK-Blue IFN-α/β cell line (InvivoGen) was maintained in HEK-293T media supplemented with 10μg/ml blasticidin (InvivoGen) and 200 μg/ml Zeocin (InvivoGen). Vero E6 cells (Cercopithecus aethiops kidney epithelial cells, ATCC CCL-81) were cultured in Minimum Eagle Medium (MEM) (11095–080, Gibco), supplemented with 2 mM L-glutamine, 50 U/mL penicillin-streptomycin and 7.5% heat-inactivated FBS. A549, BHK-21, HEK-293T and Vero E6 cell lines were originally provided by J.C. de la Torre (The Scripps Research Institute, La Jolla, CA) and HEK-Blue IFN-α/β cell line by S. Sharma (La Jolla Institute for Allergy and Immunology, La Jolla, CA)

Loureiro M.E., Zorzetto-Fernandes A.L., Radoshitzky S., Chi X., Dallari S., Marooki N., Lèger P., Foscaldi S., Harjono V., Sharma S., Zid B.M., López N., de la Torre J.C., Bavari S, & Zúñiga E. (2018). DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection. PLoS Pathogens, 14(7), e1007125.

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Evaluating NF-κB and STAT3 Pathway Modulation

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The cDNA encoding the protein ZP05614546.1 was cloned in 3xFlag (C-term) pCMV vector. Transfection was performed using transfectin™ Lipid reagent (Bio-Rad) in opti-MEM^® medium (Gibco, Life Technologies) in different epithelial cells, namely HEK293T, HT29, and TLR4/MD2/CD14 stably-transfected HEK293T (Invivogen). Different concentrations of cDNA, depending on the considered plasmid, were used: 0.8 ng.μL⁻¹ of the plasmid MAM, the plasmid containing cDNA of Carma1 and their empty equivalents, 0.08 ng.μL⁻¹ of the NF-κB reporter plasmid and 0.2 pg.μL⁻¹ of the Renilla luciferase control reporter vector. For HEK293T-TLR4/MD2/CD14, activation of the NF-κB pathway was performed by administration of LPS 100 ng.mL⁻¹ (Sigma-Aldrich). Positive control of inhibition of NF-κB pathway was obtained with SN50 50μM (Enzo Life Sciences). NF-κB reporter assay was carried out, after 24h incubation, using Dual-Luciferase^® Reporter Assay system (Promega). In the same manner, MAM activity on STAT3 pathway, activated by colivelin 0.1 nM (Santa Cruz Biotechnology), was evaluated.

Quévrain E., Maubert M.A., Michon C., Chain F., Marquant R., Tailhades J., Miquel S., Carlier L., Bermúdez-Humarán L.G., Pigneur B., Lequin O., Kharrat P., Thomas G., Rainteau D., Aubry C., Breyner N., Afonso C., Lavielle S., Grill J.P., Chassaing G., Chatel J.M., Trugnan G., Xavier R., Langella P., Sokol H, & Seksik P. (2015). Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn's disease. Gut, 65(3), 415-425.

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Cell Culture Protocols for Immunofluorescence

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HEK293T, HeLa, THP1 and THP1-blue (InvivoGen, France) cells were cultured as described in in [37] (link). For immunofluorescence, a HeLa line stably expressing EGFP-tagged NOD1 was generated. All cell lines were continuously tested for absence of mycoplasma contamination by PCR. Primary human dermal fibroblasts were obtained as previously described [38] (link).

Bielig H., Lautz K., Braun P.R., Menning M., Machuy N., Brügmann C., Barisic S., Eisler S.A., Andree M., Zurek B., Kashkar H., Sansonetti P.J., Hausser A., Meyer T.F, & Kufer T.A. (2014). The Cofilin Phosphatase Slingshot Homolog 1 (SSH1) Links NOD1 Signaling to Actin Remodeling. PLoS Pathogens, 10(9), e1004351.

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NF-κB Luciferase Assay in HEK Cell Lines

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Stable immortalized HEK 293T, HEK 293-hTLR2/6, and HEK 293/hTLR4A-MD-2-CD14 cells (InvivoGen, San Diego, CA, USA) were plated at 3 × 10⁶ cells in 6-well plates growing at 37°C in DMEM culture medium supplemented with 5% FBS, 2 mM L-glutamine, 100 U/ml gentamycin, 0.01% pyruvate, and 0.4 mM non-essential amino acids. Then, 24 h later, cells were cotransfected with the pNF3ConA Luc [nuclear factor (NF)-κB] Firefly reporter construct and the thymidine kinase promoter-Renilla reporter plasmid (100:1 ratio) using Metafectene PRO (Biontex, Plannegg, Germany) (19 (link)). Transfection medium was changed after 24 h, and cells were seeded at 1.3 × 10⁴ cells per well in 96-well plates. Then, 24 h later, ligands were added. Activities of Firefly and Renilla luciferases were measured 24 h after using TwinLite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer) in Fluo Star Optima (BMG) plate reader (three replicates per condition). All ratios were compared with the control condition (non-stimulated cells). We used FSL-1 (InvivoGen), TNF-α (InvivoGen), and LPS purified from E. coli (Sigma) and O. intermedium LPS.

Francisco S., Billod J.M., Merino J., Punzón C., Gallego A., Arranz A., Martin-Santamaria S, & Fresno M. (2022). Induction of TLR4/TLR2 Interaction and Heterodimer Formation by Low Endotoxic Atypical LPS. Frontiers in Immunology, 12, 748303.

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Cell Culture Protocols for Common Cell Lines

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LNCaP (clone FGC) (RRID:CVCL_1379), PC3 (RRID:CVCL_0035), RWPE-1 (RRID:CVCL_3791), and HEK293T (RRID:CVCL_0063) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Media and supplements were obtained from Corning Mediatech, Inc., unless otherwise noted. HEK293T cells were cultured in DMEM media supplemented with 10% v/v fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 2 mM l-alanyl-l-glutamine (Glutagro) and 1:10000 Plasmocin (Invivogen, San Diego, CA). PC3 cells were cultured in F12K media with the same supplements as above. LNCaP cells were cultured in RPMI-1640 with 1× MEM nonessential amino acids and the same supplements as above, and RWPE-1 cells were cultured in Keratinocyte SFM (Gibco 17005–042, Thermo Scientific). All cultures were used up to passage number 20 and tested monthly for Mycoplasma contamination with MycoAlert Mycoplasma detection kit (Lonza, Switzerland).

Shishodia S., Nuñez R., Strohmier B.P., Bursch K.L., Goetz C.J., Olp M.D., Jensen D.R., Fenske T.G., Ordonez-Rubiano S.C., Blau M.E., Roach M.K., Peterson F.C., Volkman B.F., Dykhuizen E.C, & Smith B.C. (2022). Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening. Journal of medicinal chemistry, 65(20), 13714-13735.

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Cultivation of Mammalian Cell Lines

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The HEK293T and HCT116 cells were obtained from ATCC (ATCC, Manassas, VA, USA). HCT116 cells with endogenous C-terminal NRF2 tagged with Nanoluc luciferase were generated in-house and have been previously described [23 (link)]. HUH-7 cells were procured from Xenotech (Kansas City, KS, USA). Both HEK293T and HUH-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and Normocin™ (Invivogen, San Diego, CA, USA) as recommended by the manufacturer to prevent bacteria, mycoplasma, and fungal contamination. HCT116-NRF2-Nanoluc cells were maintained in McCoy’s medium enriched with 10% FBS and Normocin™. All cells were cultivated in a mammalian cell incubator at 37 °C and 5% CO₂.

Li J., Arest S., Olszowy B., Gordon J., Barrero C.A, & Perez-Leal O. (2023). CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease. Antioxidants, 12(7), 1363.

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Cell Culture and Exosome Production

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Human embryonic kidney cells (HEK293T, Riken BRC), HEK-blue IFNα/β cells (InvivoGen, hkb-ifnab), and adenocarcinomic human alveolar basal epithelial cells (A549, ATCC, CCL-185) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco), supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco), 100U/ml penicillin, and 100 U/ml streptomycin (FUJIFILM Wako). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. To prepare exosome-free FCS, FCS was mixed with polyethylene glycol (PEG) 10,000 (Sigma-Aldrich) at a ratio of 5:1 and rotated at 4°C for 3 h. PEG was removed by centrifuging at 2,000 g for 20 min. The supernatant was filtered through a 0.22 µm filter and used as exosome-free FCS. The exosome-producing cell lines were cultured in advanced DMEM (Gibco) supplemented with 2% exosome-free FCS, 1nmol/L of sodium pyruvate (Nacalai Tesque), 100U/ml penicillin, and 100 U/ml streptomycin (FUJIFILM Wako).

Lyu X., Imai S., Yamano T, & Hanayama R. (2022). Preventing SARS-CoV-2 Infection Using Anti-spike Nanobody-IFN-β Conjugated Exosomes. Pharmaceutical Research, 40(4), 927-935.

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