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Hek293 cells

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HEK293 cells are a widely used cell line derived from human embryonic kidney cells. They are commonly used in research, particularly in the production and study of recombinant proteins, viral vector production, and cell-based assays.

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Lab products found in correlation

Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Bj5183 ad 1 cells, by Agilent Technologies (1 mentions) Superfect transfection reagent, by Qiagen (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Merck Group (1 mentions) Pegfp f, by Takara Bio (1 mentions) Pci neo, by Takara Bio (1 mentions) Penicillin, by Meiji Seika Pharma (1 mentions) Streptomycin, by Meiji Seika Pharma (1 mentions) Hek293 cells, by RIKEN BioResource Center (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Hela cells, by RIKEN BioResource Center (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Adeno x rapid titer kit, by Takara Bio (1 mentions) Block it u6 rnai entry vector kit, by Thermo Fisher Scientific (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Pclamp 10, by Molecular Devices (1 mentions) Digidata 1440, by Molecular Devices (1 mentions) Multiclamp 700 amplifier, by Molecular Devices (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions)

Spelling variants of this product

Lenti-X HEK293 cells (6 citations) HEK293 Tet-On 3G cells (6 citations) HEK293 Tet-On Advanced cells (3 citations) HEK293-LentiX cells (3 citations) HEK293 (2 citations)

11 protocols using hek293 cells

1

Yeast Two-Hybrid Screening of HEK293 cDNA Library

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A fresh culture of Saccharomyces cerevisiae strain PJ69-4A in YPAD media was transformed with plasmid pGBD-NS2 coding GAL4-BD-NS2 bait fusion by using the lithium acetate/polyethylene glycol (LiAc/PEG) protocol. The transformants were selected on synthetic dropout (SD) agar plates (without Trp) and checked for the NS2 gene with PCR. One of the bait colonies was grown in YPAD media and retransformed with a cDNA library derived from HEK293 cells (Clontech, #638826), and screened following the matchmaker two-hybrid system protocol. The transformants were selected on SD agar plates (without His, Leu, Ade, and Trp) for the primary screening, and then tested with the β-galactosidase assay for the second screening. The plasmids carrying the cDNAs were isolated from potential transformants and confirmed to be positive by at least 2 independent tests with a yeast plasmid DNA miniprep kit (Bio Basic, Markham, ON, Canada) according to the manufacturer’s instructions. The plasmid samples were transformed in E. coli DH5α and were amplified. The cDNA inserts in the plasmids were sequenced and identified with BLAST(Basic Local Alignment Search Tool) analysis.
ŞENBAŞ AKYAZI B., PİRİNÇAL A., KAWAGUCHI A., NAGATA K, & TURAN K. (2020). Interaction of influenza A virus NS2/NEP protein with the amino-terminal part of Nup214. Turkish Journal of Biology, 44(2), 82-92.
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2

Adenoviral Generation of HKDC1 Overexpression

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Adenovirus containing the human HKDC1 cDNA was generated as previously described (21 ). In brief, fragment containing the 2,750 bp ORF of HKDC1 (RC221178 plasmid, Origene, Rockville, MD, USA), was cloned into the pShuttle-CMV adenoviral vector (Agilent, Santa Clara, CA, USA). The resulting HKDC1 adenoviral shuttles were then linearized and transformed into BJ5183-AD1 cells (Agilent) producing recombinant clones which were isolated and digested. Recombinant HKDC1 adenoviruses were produced by transfecting HEK293 cells (Clontech, Mountainview, CA, USA) with PacI-digested recombinant plasmid DNA and FuGene36. After transfection was complete, the viral lysate was collected and further amplified in HEK293 cells. Infected cells were collected and lysed by two freeze/thaw cycles in 2 ml freeze/thaw buffer (lOmM Tris/HCl, pH 8.0, ImM MgC12). The virus was then purified by ultracentrifugation using a CsCl gradient. Purified virus was de-salted using a 7k MWCO column (Thermo Scientific, Waltham, MA, USA) and equilibrated with freeze/thaw buffer, after which glycerol was added to a final concentration of 10%. Viruses were titrated by measuring OD260 (1 OD260 = 1.1 Å~ 1012 virions ml-1) and by plaque assay in HEK293 cells.
Khan M.W., Priyadarshini M., Cordoba-Chacon J., Becker T.C, & Layden B.T. (2018). Hepatic hexokinase domain containing 1 (HKDC1) improves whole body glucose tolerance and insulin sensitivity in pregnant mice. Biochimica et biophysica acta. Molecular basis of disease, 1865(3), 678-687.
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3

HEK293 Cell Transfection and Culture

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HEK293 cells (RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan, RCB Cat# RCB1637, RRID: CVCL_0045) were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, United States) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, United States), 30 U mL−1 penicillin (Meiji Seika Pharma, Tokyo, Japan), and 30 μg mL−1 streptomycin (Meiji Seika Pharma). HEK293 cells (with a density of 100,000/well) were co-transfected with the vector 1.0 μg pCI-neo or 1.0 μg pCI-neo-mouse TRPV1 and 0.1 μg pEGFP-F (Clontech, Mountain View, CA, United States) as a marker. Transfection was carried out using SuperFect Transfection Reagent (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. HEK293 cells were trypsinized and plated onto glass coverslips 18 h after transfection. Then cells were subjected to measurements 12–42 h after plating on the coverslips.
Takahashi K., Araki K., Miyamoto H., Shirakawa R., Yoshida T, & Wakamori M. (2021). Capsaicin and Proton Differently Modulate Activation Kinetics of Mouse Transient Receptor Potential Vanilloid-1 Channel Induced by Depolarization. Frontiers in Pharmacology, 12, 672157.
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4

Cell Culture Maintenance Protocol

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HeLa cells were obtained from RIKEN BRC. HEK293 cells were purchased from Clontech. NSC34 cells were provided by Dr. Neil Cashman.68 (link) These cells were maintained at 37°C with 5% CO2 in a humidified incubator. Both cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 units/mL) (Sigma, P4333). These cell lines were periodically confirmed to be free of mycoplasma.
Miyagi T., Ueda K., Sugimoto M., Yagi T., Ito D., Yamazaki R., Narumi S., Hayamizu Y., Uji-i H., Kuroda M, & Kanekura K. (2023). Differential toxicity and localization of arginine-rich C9ORF72 dipeptide repeat proteins depend on de-clustering of positive charges. iScience, 26(6), 106957.
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5

Rat Dental Epithelial and HEK293 Cell Culture

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Rat dental epithelial cell line, HAT-7 cells [15 (link)] and human embryonic kidney (HEK) 293 cells (Clontech) were maintained in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12, Life Technologies) and DMEM (Life Technologies), respectively.
Govitvattana N., Kaku M., Ohyama Y., Jaha H., Lin I.P., Mochida H., Pavasant P, & Mochida Y. (2021). Molecular cloning of mouse homologue of enamel protein C4orf26 and its phosphorylation by FAM20C. Calcified tissue international, 109(4), 445-454.
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6

Liver-Selective Retsat Depletion in Mice

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Adenoviral vectors expressing shRNA against β-Galactosidase (βGal) or RetSat were cloned using the BLOCK-iT U6 RNAi Entry Vector Kit and Adenoviral RNAi Expression System according to the manufacturer’s instructions (ThermoFisher). Adenoviruses expressing GFP or a GFP- murine ChREBP fusion protein are described elsewhere25 (link). Viruses were amplified in HEK293 cells (Clontech) and purified by standard CsCl gradient centrifugation and dialyzed against 0.9% saline. Titers were determined by the Adeno-X Rapid Titer Kit (Clontech). For liver-selective RetSat depletion, ~10E10 infectious units of shβGal-or shRetSat-expressing adenoviruses were injected via the tail vein into lean or obese, insulin-resistant mice.
Heidenreich S., Witte N., Weber P., Goehring I., Tolkachov A., von Loeffelholz C., Döcke S., Bauer M., Stockmann M., Pfeiffer A.F., Birkenfeld A.L., Pietzke M., Kempa S., Muenzner M, & Schupp M. (2017). Retinol saturase coordinates liver metabolism by regulating ChREBP activity. Nature Communications, 8, 384.
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7

Transient Co-transfection of HEK293 Cells

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Human embryonic kidney HEK293 cells (QBiogene) were transiently
co-transfected with a 2:1 mixture of Nav1.7-expressing
plasmid8 (link) and a
pIRES2-ZsGreen bicistronic plasmid (Clontech) expressing ZsGreen and mouse FHF2B
proteins10 (link). The same
pIRES2-ZsGreen plasmid without FHF2 coding sequence served as control. Cells
were trypsinized 3 hours post-transfection, seeded onto gelatinized coverslips,
and were used for recording after 48 hr. For sodium current recordings,
coverslips were transferred to recording chamber containing carbogen-buffered
bath solution (115 mM NaCl, 26 mM NaHCO3, 3 mM KCl, 10 mM glucose, 4
mM MgCl2, 2 mM CaCl2, 0.2 mM CdCl2, 3 mM
myoinositol, 2 mM Na pyruvate, 7 mM NaOH-buffered HEPES pH 7.2) at 25 °C
and green fluorescent cells were whole-cell patched with pipettes filled with
104 mM CsF, 50 mM tetraethylamine chloride, 10 mM HEPES pH 7.2, 5 mM glucose, 2
mM MgCl2, 10 mM EGTA, 2 mM ATP, 0.2 mM GTP and having 1–2
MΩ resistance. All voltage commands and current recordings were made
using a MultiClamp 700 amplifier, digital/analog converter Digidata 1440, and
pClamp10 software (Molecular Devices – Axon Instruments). For all
recording protocols, voltage-gated current was isolated during data acquisition
by P/N subtraction of leak and capacitive currents (N = −6).
Marra C., Hartke T.V., Ringkamp M, & Goldfarb M. (2022). Enhanced sodium channel inactivation by temperature and FHF2 deficiency blocks heat nociception. Pain, 164(6), 1321-1331.
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8

HEK293 Cell Culture Protocols

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HEK293 cells (Clontech Laboratories) were used for most experiments described in this study. For the enzymatic activity assay, HEK293T cells from American Type Culture Collection were used. HEK293 Cosmc-KO cells (referred to as SimpleCells throughout the text) and their unaltered counterpart were obtained from the laboratory of Prof. Henrik Clausen (Copenhagen Center for Glycomics, Denmark). All cell lines were maintained in a growth medium with high glucose (4500 mg/L) supplemented with 10% FBS and either 100 U/ml of Antibiotic Antimycotic or 10 mg/ml of Penicillin-Streptomycin in humidified atmosphere with 5% CO2 at 37 °C.
Gravdal A., Xiao X., Cnop M., El Jellas K., Johansson S., Njølstad P.R., Lowe M.E., Johansson B.B., Molven A, & Fjeld K. (2021). The position of single-base deletions in the VNTR sequence of the carboxyl ester lipase (CEL) gene determines proteotoxicity. The Journal of Biological Chemistry, 296, 100661.
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9

Transient Co-Expression of Nav1.7 and FHF2B in HEK293 Cells

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Human embryonic kidney HEK293 cells (QBiogene) were transiently cotransfected with a 2:1 mixture of Nav1.7-expressing plasmid8 (link) and a pIRES2-ZsGreen bicistronic plasmid (Takara Bio, San Jose, CA) expressing ZsGreen and mouse FHF2B proteins.10 (link) The same pIRES2-ZsGreen plasmid without FHF2 coding sequence served as the control. Cells were trypsinized 3 hours posttransfection, seeded onto gelatinized coverslips, and were used for recording after 48 hours. For sodium current recordings, coverslips were transferred to recording the chamber containing carbogen-buffered bath solution (115 mM NaCl, 26 mM NaHCO3, 3 mM KCl, 10 mM glucose, 4 mM MgCl2, 2 mM CaCl2, 0.2 mM CdCl2, 3 mM myoinositol, 2 mM Na pyruvate, 7 mM NaOH-buffered HEPES, and pH 7.2) at 25°C and green fluorescent cells were whole-cell patched with pipettes filled with 104 mM CsF, 50 mM tetraethylamine chloride, 10 mM HEPES pH 7.2, 5 mM glucose, 2 mM MgCl2, 10 mM EGTA, 2 mM ATP, and 0.2 mM GTP and having 1 to 2 MΩ resistance. All voltage commands and current recordings were made using a MultiClamp 700 amplifier, digital or analog converter Digidata 1440, and pClamp10 software (Molecular Devices, San Jose, CA). For all recording protocols, voltage-gated current was isolated during data acquisition by P/N subtraction of leak and capacitive currents (N = −6).
Marra C., Hartke T.V., Ringkamp M, & Goldfarb M. (2022). Enhanced sodium channel inactivation by temperature and FHF2 deficiency blocks heat nociception. Pain, 164(6), 1321-1331.
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10

Generating Polyclonal DGN Antibodies

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Polyclonal DGN antibody Sheep173 was made by immunizing a sheep with a His-tagged rabbit DGN protein (amino acid 1–315 of α-DG) grown in a stable mammalian cell line [HEK293 cells; American Type Culture Collection (ATCC)] and purified by Talon beads (Takara Bio USA, Inc.). The anti-DGN antibodies in the sheep serum were affinity purified by affinity strips containing rabbit DGN purified from E. coli and transferred to a polyvinylidene difluoride membrane (Immobilon FL-Membrane; Millipore) to enrich antibodies to α-DGN lacking glycans and, thus, exclude any carbohydrate-specific antibody epitopes. Polyclonal α-DG antibody Sheep174 targeting the mucin region of α-DG (amino acid 316–485 of α-DG) was made by immunizing a sheep with a His-tagged protein grown in a stable mammalian cell line (HEK293 cells; ATCC) and purified by Talon beads (Takara Bio USA, Inc.). The anti-α-DG antibodies to the mucin region were affinity purified by affinity strips containing GST-mucin α-DG purified from E. coli and transferred to a polyvinylidene difluoride membrane (Immobilon FL-Membrane; Millipore). Using E. coli protein of the mucin α-DG region for affinity purification ensured we enriched for antibodies to α-DG lacking glycans in the mucin region and excluded any carbohydrate-specific antibody epitopes.
de Greef J.C., Slütter B., Anderson M.E., Hamlyn R., O’Campo Landa R., McNutt E.J., Hara Y., Pewe L.L., Venzke D., Matsumura K., Saito F., Harty J.T, & Campbell K.P. (2019). Protective role for the N-terminal domain of α-dystroglycan in Influenza A virus proliferation. Proceedings of the National Academy of Sciences of the United States of America, 116(23), 11396-11401.
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