Hek293t cells

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, China, Germany, Canada, Japan, Australia

HEK293T cells are a widely used human embryonic kidney cell line. They are a transformed cell line that can be used for a variety of laboratory applications, including protein expression, virus production, and cell-based assays.

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361 protocols using hek293t cells

Trogocytic Assay for SARS-CoV-2 Spike Antibodies

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HEK-293T cells (Invitrogen) were transfected using SARS-CoV-2 S expression plasmid expression vector and lipofectamine (thermofisher) in OptiMEM as previously described.⁸⁶ (link) SARS-CoV-2 S expression HEK293T cells were stained with PKH26 dye according to the manufactures protocol (Sigma-Aldrich). Next, the stained HEK293T cells were incubated for 30 min at 37°C with serial antibodies dilutions. 2G12-IgG1, specific for HIV-1 gp120, was used as a negative control. After incubation, the cells were washed and THP-1 cells (ATCC), stained with carboxyfluorescein succinimidyl ester according to manufactures protocol (Thermofisher), were added to the HEK293T cells at a 2:1 effector:target cell ratio. The plates were spun down for 30 sec to promote cell to cell contact before incubation for 1 hours at 37°C. After incubation, the plates were washed twice, resuspended in PBS 2% fetal calf serum (FCS) and analyzed using flow cytometry. Trogocytic activity was calculated by the mean fluorescence intensity (MFI) of the double positive PKH26+ CFSE+, THP-1 cells and depicted as the area under the curve.

Radić L., Sliepen K., Yin V., Brinkkemper M., Capella-Pujol J., Schriek A.I., Torres J.L., Bangaru S., Burger J.A., Poniman M., Bontjer I., Bouhuijs J.H., Gideonse D., Eggink D., Ward A.B., Heck A.J., Van Gils M.J., Sanders R.W, & Schinkel J. (2023). Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses. iScience, 26(4), 106540.

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Dual Luciferase Assay for miR-99a Targeting

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Gene prediction software (https://cm.jefferson.edu/rna22/Precomputed/) was adopted to predict the target gene ofmiR-99a. The 3’UTR fragments of BRD4 containing both mutant (MUT) and wild-type (WT) binding sites of miR-99a were amplified by polymerase chain reation (PCR) and cloned into the vector pMIR-REPORLuciferase (Promega, Madison, WI, United States of America) to form luciferase reporter vectors. HEK-293T cells (Invitrogen Life Technologies, Carlsbad, CA, United States of America) were spread on 96-well plates at 4 × 103 (link) cells/well. HEK-293T cells were transfected withmiR-99a mimic + BRD4-wild type (WT), miR-99a mimic + BRD4-mutant type (MUT), mimic NC + BRD4-WT, or mimic NC + BRD4-MUT with lipofectamine 2000 (Invitrogen). At 48 h after transfection, cells were harvested, and luciferase activity was measured using dual luciferase reporter gene kit (Promega, Madison, WI, United States of America). The ratio of firefly luciferase activity to Renilla luciferase activity indicated the relative activity of luciferase21 (link).

Bie X., Ao J, & Zhu D. (2023). Sevoflurane attenuates myocardial ischemia/reperfusion injury by up-regulating microRNA-99a and down-regulating BRD4. Acta Cirúrgica Brasileira, 38, e383123.

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Cell Culture and Lentiviral Transduction

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A549 cells derived from human lung cancer and HEK-293T cells were obtained from China Center for Type Culture Collection (Wuhan, China). The A549 cells and HEK-293T cells were maintained in Ham’s F12/K medium and Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen, Carlsbad, CA, USA), respectively, supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Carlsbad, CA, USA), 2 mM L-glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin at 37 °C in a humidified 5% CO₂. For lentivirus infection, A549 cells (5 × 10⁴ cells/well) were seeded on 24-well plates for 12 h, infected by lentivirus (MOI = 20) with 5 μg/ml of polybrene for another 12 h according to the manufacturer’s protocol and then cultured in F12/K medium containing 10% FBS for 72 h. The efficiency of virus infection was observed under fluorescence-inverted microscope (Olympus, Japan). For transfection of plasmid, plasmid DNAs were transiently transfected into A549 cells or HEK-293T cells with Lipofectamine 2000 according to the manufacturer’s protocols (Invitrogen, Carlsbad, CA, USA).

Zhao M., Li Y., Guo C., Wang L., Chu H., Zhu F., Li Y., Wang X., Wang Q., Zhao W., Shi Y., Chen W, & Zhang L. (2018). IL-37 isoform D downregulates pro-inflammatory cytokines expression in a Smad3-dependent manner. Cell Death & Disease, 9(6), 582.

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Purification of EGFP-Myo19 from HEK293T

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A nonmuscle-type myosin light chain (MRLC12A) cDNA was obtained from a human kidney cDNA library and transferred to pEB Multi vector (Fujifilm Wako Chemicals). HEK293T cells (Invitrogen) were transfected with the vector DNA, and neomycin-resistant MRLC12A-expressing HEK293T cells were created. EGFP-HM19^FullLZ was then transiently expressed in MRLC12A-expressing HEK293T cells by a transfection of recombinant Myo19 heavy chain cDNAs using PEI Max (Polysciences, Inc) and then purified using anti-FLAG antibody-agarose resins (SIGMA). In short, 6 × 10⁷ cells were harvested at 24 h after transfection and homogenized in a lysis buffer (0.25 M KCl, 50 mM Hepes–KOH [pH 7.5], 5 mM EGTA, 15 mM β-mercaptoethanol, 1 mM ATP, 2% CHAPS, and protease inhibitor cocktail [described previously]). The suspension was mixed with anti-FLAG antibody-agarose resins, and the tube containing the suspension was rotated at 4 °C for 1 h. After two washes with a wash buffer (0.12 M KCl, 12.5% trehalose, 1 mM EGTA, 15 mM β-mercaptoethanol, 2% CHAPS, and protease inhibitor cocktail), the protein was eluted with an elution buffer (the wash buffer containing 0.1 mg/ml FLAG peptide). The eluted fractions were flash-frozen by liquid nitrogen and stored at −80 °C.

Sato O., Sakai T., Choo Y.Y., Ikebe R., Watanabe T.M, & Ikebe M. (2022). Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells. The Journal of Biological Chemistry, 298(5), 101883.

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Cell Culture Conditions for Transcriptional Activation and Nuclease Specificity Experiments

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All cells were cultured at 37 °C in a 5% CO₂ atmosphere. HEK293T cells (Life Technologies) used in transcriptional activation experiments were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (CellGro) supplemented with 10% FBS (CellGro) and 1% penicillin–streptomycin–glutamine (CellGro). HEK293T cells used in surveyor assays and nuclease specificity experiments and U2OS.eGFP-PEST cells^{26 (link)} stably integrated with an eGFP-PEST fusion gene were maintained in DMEM (Life Technologies) supplemented with 10% FBS, 1× penicillin–streptomycin–glutamax (Life Technologies) and 400 μg/mL of the selection antibiotic G418 (for the U2OS. eGFP-PEST cells). Cells were continuously maintained at <90% confluency. All cell lines were sourced commercially or were functionally validated. Cells were periodically tested for mycoplasma contamination using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza).

Maji B., Moore C.L., Zetsche B., Volz S.E., Zhang F., Shoulders M.D, & Choudhary A. (2016). Multidimensional chemical control of CRISPR–Cas9. Nature chemical biology, 13(1), 9-11.

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Cell Line Characterization and Culture

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SMA-497, SMA-540 and SMA-560 cells were kindly provided by Dr. D. D. Bigner (Durham, NC) and GL-261 cells were obtained from ATCC (Rockville, MD). Human embryonic kidney (HEK) 293T cells were purchased from Open BioSystems (Huntsville, AL). Cells were grown as adherent monolayers in Dulbecco´s modified Eagle´s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine (Gibco, Waltham, MA). Cells were regularly tested for mycoplasma contamination using the enzymatic MycoAlert^™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

Quijano-Rubio C., Silginer M, & Weller M. (2022). CRISPR/Cas9-mediated abrogation of CD95L/CD95 signaling-induced glioma cell growth and immunosuppression increases survival in murine glioma models. Journal of Neuro-Oncology, 160(2), 299-310.

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HEK293T Cell Transfection Protocol

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HEK293T cells were purchased from ATCC (Manassas, VA) and maintained in complete RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS). HEK293T cells were transfected with Arrest-IN reagent (Open Biosystems) at a 6:1 ratio with DNA (μl:μg) using 2–6 μg of plasmid DNA. Ringer’s solution was obtained from the Veterinary Pharmacy at the NIH. All experiments were conducted in accordance with institutional guidelines.

Prickett T.D., Zerlanko B.J., Hill V.K., Gartner J.J., Qutob N., Jiang J., Simaan M., Wunderlich J., Gutkind J.S., Rosenberg S.A, & Samuels Y. (2014). Somatic mutation of GRIN2A in malignant melanoma results in loss of tumor suppressor activity via aberrant NMDAR complex formation. The Journal of investigative dermatology, 134(9), 2390-2398.

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Cell Culture of MDA-MB-231 and HEK-293T

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MDA-MB-231 were obtained from ATCC (Lot: 5883183, cultures initiated 19/08/2010) and maintained in RPMI 1640 (Life Technologies, Paisley, UK) supplemented with 10% FBS (PAA Laboratories Ltd., Somerset, UK). HEK-293T cells were obtained from Open Biosystems (Lot: L0708; Thermo Scientific, Leicestershire, UK) and maintained in DMEM + 10% FBS. All studies were conducted using early passage cultures typically within 3 months of thawing from cell stocks confirmed mycoplasma negative.

Singleton D.C., Rouhi P., Zois C.E., Haider S., Li J.L., Kessler B.M., Cao Y, & Harris A.L. (2014). Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis. Oncogene, 34(36), 4713-4722.

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Lentiviral shRNA Knockdown Validation

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Verified shRNA clones were obtained from the Open Biosystems pGIPz library (Victorian Centre for Functional Genomics). shRNA-expressing lentiviral plasmids were transfected using Lenti-X packaging vectors into HEK293T cells (Open Biosystems). Viral containing media was collected, filtered and stored at − 80 °C. Target cells were transduced and selected using puromycin. Knockdown was confirmed using quantitative real-time PCR and Western blot.

Busuttil R.A., George J., House C.M., Lade S., Mitchell C., Di Costanzo N.S., Pattison S., Huang Y.K., Tan P., Cheong J.H., Rha S.Y, & Boussioutas A. (2020). SFRP4 drives invasion in gastric cancer and is an early predictor of recurrence. Gastric Cancer, 24(3), 589-601.

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Lentiviral Pseudotyping with VSV-G

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Viral stocks pseudotyped with the vesicular stomatitis G protein (VSV-G) were prepared by transient co-transfection of HEK293 T cells (Open Biosystems) using the ViraPower™ Lentiviral Packaging Mix (Invitrogen) following manufacturer’s instructions. Supernatant was collected 48 h post transfection and passed through a 0.45 μm syringe filter. Lentivirus was concentrated by ultracentrifugation using a Beckman SW28 swinging bucket rotor at 19,500 rpm for 2.5 h at 16°C, reconstituted in PBS, and stored at −80°C until use.

Tisdale S., Van Alstyne M., Simon C.M., Mentis G.Z, & Pellizzoni L. (2022). SMN controls neuromuscular junction integrity through U7 snRNP. Cell reports, 40(12), 111393.

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