Hek293 cells

Manufactured by Merck Group

Sourced in United States, Germany

HEK293 cells are a commonly used human embryonic kidney cell line. They are a type of adherent cell that can be cultured in vitro. HEK293 cells are widely used in various research applications, including the production of recombinant proteins, viral vector production, and the study of cell signaling pathways.

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HEK293 (51 citations) HEK293 cell line (6 citations) HEK293-APP695 cells (2 citations)

43 protocols using hek293 cells

HEK293 Cell Culture Protocol

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HEK293 cells (female, SIGMA) were maintained in DMEM (SIGMA) media supplemented with 10% fetal bovine serum (SIGMA) and 4 mM GlutaMAX (Gibco) at 37°C and 5% CO₂. HEK293-6E were grown in suspension in serum-free F17 culture media (Invitrogen) supplemented with 0.1% Kolliphor P188 (SIGMA) and 4 mM GlutaMAX. Cultures were grown at 37°C and 5% CO₂ under constant agitation (120 rpm).

Narimatsu Y., Joshi H.J., Nason R., Van Coillie J., Karlsson R., Sun L., Ye Z., Chen Y.H., Schjoldager K.T., Steentoft C., Furukawa S., Bensing B.A., Sullam P.M., Thompson A.J., Paulson J.C., Büll C., Adema G.J., Mandel U., Hansen L., Bennett E.P., Varki A., Vakhrushev S.Y., Yang Z, & Clausen H. (2019). An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells. Molecular cell, 75(2), 394-407.e5.

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Lentiviral Packaging Protocol for HEK293 Cells

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HEK293 cells (American Type Culture Collection, 2.5 × 10⁶ per well) were transfected on a 1% PEI (Sigma Aldrich; Cat #P3143) coated six-well plate in 2.5 mL DMEM (Invitrogen, Cat #11965118) with 10% FBS (Phoenix Scientific, Cat #PS-100-02-500) according to the Lipofectamine 3000 protocol (Thermo Fisher Scientific, Cat #L3000015). Briefly, psPAX (Addgene Cat #12260; 0.8 μg), pMD302 (Addgene Cat #12259; 0.3 μg), and pAdvantage (Promega Cat #E1711; 0.1 μg) viral packaging plasmids were added to P3000 reagent with 1.2 μg plasmid (MyoD – vector ID #VB200213-1024pkg or Tet3g – vector ID #VB200811-1097xwy) before adding dropwise to cells.
The next day, media was replaced on HEK cells with 3 mL fresh medium supplemented with 6 μL 500× ViralBoost reagent (ALSTEM, Cat #VB100). On day 4, virus was harvest by pooling supernatants and centrifuged for 10 min at 500×g. Clarified supernatant was transferred to a new tube and Lenti-X concentrator (Takara, Cat #631231) was added at a 1:3 ratio (concentrator:media). The supernatant was incubated at 4 °C for 45 min with Lenti-X concentrator and centrifuged at 1,500×g for 45 min at 4 °C. Supernatant was removed, and pellet resuspended in media at 1/10 original volume.

Geist Hauserman J., Laverty C.G., Donkervoort S., Hu Y., Silverstein S., Neuhaus S.B., Saade D., Vaughn G., Malicki D., Kaur R., Li Y., Luo Y., Liu P., Burr P., Foley A.R., Mohassel P, & Bönnemann C.G. (2024). Clinical, immunohistochemical, and genetic characterization of splice-altering biallelic DES variants: Therapeutic implications. Human Genetics and Genomics Advances, 5(2), 100274.

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Recombinant Factor B Mutagenesis

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Plasmids containing HIS-tagged CFB were generously gifted by Alexion Pharmaceuticals, Inc. Mutagenesis (CFB c.1101C>G) was performed using the QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Inc). All constructs were sequenced confirmed. Recombinant proteins (mutant FB p.Arg367 and WT FB p.Ser367) were produced in HEK293 cells (Sigma-Aldrich, Inc) and purified using the HisPur™ Ni-NTA purification kit (Thermo Fisher Scientific, Inc).

Zhang Y., Kremsdorf R., Sperati C.J., Henriksen K.J., Mori M., Goodfellow R.X., Pitcher G.R., Benson C., Borsa N.G., Taylor R.P., Nester C.M, & Smith R.J. (2020). Mutation of complement factor B causing massive fluid-phase dysregulation of the alternative complement pathway can result in atypical hemolytic uremic syndrome. Kidney international, 98(5), 1265-1274.

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Culturing Human Monocytic and HEK293 Cells

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THP-1 cells (human acute monocytic leukemia, DSMZ, Braunschweig, Germany) were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS, PAN-Biotec GmbH, Aidenbach, Germany), and 1% penicillin streptomycin (P/S, Sigma-Aldrich, Darmstadt, Germany) by seeding at 2.0×10⁵ cells/ml and subculture at 1×10⁶ cells/ml. HEK293 cells (ATCC CRL-1573^TM) were cultured in DMEM high glucose (Sigma-Aldrich) supplemented with 10% FBS and 1% P/S. Transfected and transduced cells were cultured in medium supplemented with additional 1 mg/ml geneticin (G418, Calbiochem EMD Chemicals, San Diego, USA) for selection. Prior to experiments, transduced cells were cultured without G418 for at least one week.

Koenen A., Babendreyer A., Schumacher J., Pasqualon T., Schwarz N., Seifert A., Deupi X., Ludwig A, & Dreymueller D. (2017). The DRF motif of CXCR6 as chemokine receptor adaptation to adhesion. PLoS ONE, 12(3), e0173486.

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Antibody Production in Cell Lines

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THP-1 monocytic cells (Sigma Aldrich, 88081201-1VL) were cultured in RPMI with 10% FBS and L-glutamine and were kept at 2.5-10 × 10⁵ cells per mL. HEK293 cells (Sigma Aldrich, 12022001‐1VL) and Expi293F (Thermofisher, A14527) cells were used to produce antibodies similarly as done before^{37 (link),38 (link)}. Supernatant containing produced antibodies was purified through incubation with protein G sepharose 4 fast flow (Cytiva, 17-0618-05) according to the manufacturer’s instruction.

Izadi A., Karami Y., Bratanis E., Wrighton S., Khakzad H., Nyblom M., Olofsson B., Happonen L., Tang D., Sundwall M., Godzwon M., Chao Y., Toledo A.G., Schmidt T., Ohlin M., Nilges M., Malmström J., Bahnan W., Shannon O., Malmström L, & Nordenfelt P. (2024). The hinge-engineered IgG1-IgG3 hybrid subclass IgGh47 potently enhances Fc-mediated function of anti-streptococcal and SARS-CoV-2 antibodies. Nature Communications, 15, 3600.

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Structural Characterization of Recombinant and Plant-Produced hOPN

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Circular dichroism (CD) spectra were recorded by Chirascan (Applied Photophysics, Ltd) to determine the secondary structure of recombinant hOPN expressed in HEK 293 cells (Sigma-Aldrich, USA) and plant-produced hOPN. The spectra were measured between 190 nm and 250 nm. The measurements were conducted using protein concentrations of 0.10 mg/mL in 10 mM potassium phosphate buffer (pH 7.4). All data presented are the means of three independent measurements. The secondary contents of both proteins were calculated using Raussens et al. method⁴⁶.

Rattanapisit K., Abdulheem S., Chaikeawkaew D., Kubera A., Mason H.S., Ma J.K., Pavasant P, & Phoolcharoen W. (2017). Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells. Scientific Reports, 7, 17358.

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Imaging of CASK Aggregation in HEK-293 Cells

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Human embryonic kidney (HEK-293) cells (ATCC) were plated on 50 µg/ml poly-L-lysine (Sigma Aldrich Inc.)-coated coverslips (Fisherbrand, Inc.) in 24-well plates (JetBiofil) and maintained in DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone) supplemented with 5 mg/ml penicillin-streptomycin (Hyclone). Cells at 80% confluency were transfected with 0.5 µg of GFP-CASK-WT and GFP-CASK^L209P DNA per well using the calcium phosphate method. Twenty hours post-transfection, cells were washed twice with phosphate buffered saline (Sigma Inc.) and fixed for 15 minutes at room temperature using a 4% paraformaldehyde solution. Coverslips were mounted on microscope slides (Premiere) using Vectashield (Vector Laboratories Inc.) and visualized using confocal laser scanning microscopy (ZEISS Axio Examiner.Z1 LSM 710). The percentage of transfected cells with visible aggregates was counted using the cell counter plugin of the Image J program. For each condition, five high-power field images were analyzed for aggregation. Total cells and cells containing aggregates were visually identified and tallied. Percent of total cells containing aggregates was then calculated. The image analysis procedure was repeated 3 times for each condition and averaged.

LaConte L.E., Chavan V., DeLuca S., Rubin K., Malc J., Berry S., Summers C.G, & Mukherjee K. (2018). An N-terminal heterozygous missense CASK mutation is associated with microcephaly and bilateral retinal dystrophy plus optic nerve atrophy. American journal of medical genetics. Part A, 179(1), 94-103.

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Cell Culture Conditions for Various Cell Lines

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HEK293 cells (#85120602, Sigma), HeLa (CCL-2, ATCC) and AGS cells (CRL-1739, ATCC) were maintained in DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma) and 4 mM GlutaMAX (Gibco). MCF7 (HTB-22, ATCC) were cultured in DMEM low glucose (Gibco) containing 10% fetal bovine serum, 4 mM GlutaMAX and 10 µg/mL insulin (Gibco). SH-SY5Y (CRL-2266, ATCC) cells were maintained in 50% RPMI-1640 medium (Sigma), 50% DMEM low glucose, supplemented with 10% fetal bovine serum and 4 mM GlutaMAX. Suspension HEK293-6E cells^{95 (link)} were grown in serum-free Freestyle F17 (Gibco) supplemented with 0.1% Kolliphor P188 (Sigma) and 4 mM GlutaMAX under constant agitation (120 rpm). CHOZN GS–/– cells (Sigma) were grown in 50% BalanCD CHO growth medium (Irvine Scientific) and 50% Ex-Cell CD medium (Sigma) supplemented with 8 mM GlutaMAX. All cells were cultured in humidified incubators at 37°C and 5% CO_2.

Sørensen D.M., Büll C., Madsen T.D., Lira-Navarrete E., Clausen T.M., Clark A.E., Garretson A.F., Karlsson R., Pijnenborg J.F., Yin X., Miller R.L., Chanda S.K., Boltje T.J., Schjoldager K.T., Vakhrushev S.Y., Halim A., Esko J.D., Carlin A.F., Hurtado-Guerrero R., Weigert R., Clausen H, & Narimatsu Y. (2023). Identification of global inhibitors of cellular glycosylation. Nature Communications, 14, 948.

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Culturing Transformed and Primary Cells

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tsA201 cells are transformed HEK293 cells (Sigma-Aldrich). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin and subcultured every 3–4 d using 0.05% trypsin containing EDTA. Nontransformed pancreatic duct epithelial cells (PDECs), a cell line originally derived from the main pancreatic duct of a dog (Oda et al., 1996 (link)), were cultured on Transwell inserts (Corning) coated with collagen (Advanced BioMatrix Inc.) over a feeder layer of confluent human gallbladder myofibroblasts (Oda et al., 1996 (link)). For intracellular Ca²⁺ measurement, the single PDECs were plated on 5-mm small glass chips coated with Vitrogen. The tsA201 cells were plated on small chips coated with poly-l-ornithine for Ca²⁺ imaging and confocal experiments. All experiments were performed at room temperature (22–24°C).

Jung S.R., Seo J.B., Deng Y., Asbury C.L., Hille B, & Koh D.S. (2016). Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling. The Journal of General Physiology, 147(3), 255-271.

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Codon-optimized CHT and LV-AA mutant expression

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Sequences corresponding to full-length CHT (NM_021815.4) and LV-AA mutation at residues 531–532 (Ribeiro et al., 2005 (link); Ruggiero et al., 2012 (link)) were codon-optimized for expression in human cell lines and custom-synthesized by GeneArt (LifeTech). Constructs were designed with a N-terminal FLAG epitope (Cuddy et al., 2012 (link)) and cloned into the pLenti6.3/V5 DEST vector which also contained a C-terminal V5 epitope tag (LifeTech). Lentiviral particles were generated (ViraPower, Thermofisher Scientific) and used to transduce HEK-293 cells (Sigma Aldrich). Pools of stable transformants were selected with 8 μg/mL Blasticidin. In addition, SH-SY5Y cells (ATCC) were transduced with a CHT construct tagged with GFP at the C-terminal (Origene, PS100071) or a FAP tag at the N-terminus (Sharp Edge Laboratory). Both stable pools and single clones were expanded. HEK-293 cells were cultured in DMEM High glucose (Gibco # 21969-035) supplemented with 10% FBS and 4 mM Glutamine (Thermofisher Scientific). SH-SY5Y cells were cultured in DMEM:F12 with Glutamine (Gibco # 11320-033) supplemented with 15% FBS and 1x NEAA (Thermofisher Scientific).

Choudhary P., Armstrong E.J., Jorgensen C.C., Piotrowski M., Barthmes M., Torella R., Johnston S.E., Maruyama Y., Janiszewski J.S., Storer R.I., Skerratt S.E, & Benn C.L. (2017). Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter. Frontiers in Molecular Neuroscience, 10, 40.

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