Hek293 cells

Manufactured by Corning

Sourced in United States

HEK293 cells are a type of immortalized human embryonic kidney cells commonly used in cell and molecular biology research. They are derived from human embryonic kidney cells transformed with sheared human adenovirus type 5 DNA. HEK293 cells are a widely utilized cell line for various experimental applications, including protein expression, virus production, and toxicology studies.

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11 protocols using hek293 cells

Transient Transfection of HEK293 Cells

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HEK293 cells from the American Type Culture Collection were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Cellgro) with 10% fetal bovine serum (FBS, HyClone) at 37°C, 5% CO₂. For radioligand binding, fluorescence, cAMP, and BRET assays the cells were plated in six-well plates (5×10⁵ cells/well) 24hrs prior to transfection. For confocal imaging, BiFC, and FCS cells were plated on poly-lysine treated glass coverslips in six-well plates. Transfections contained 100ng of each plasmid with 2ul of lipofectamine reagent (Invitrogen), unless otherwise indicated. Transfections were performed in serum-free DMEM and were ended after 5hrs by washing in PBS and addition of DMEM. For confocal, BiFC, and FCS transfections were ended in phenol-free MEM. Assays were performed 24hrs following transfection. Transfection efficiency was 40%, determined by confocal fluorescence imaging of cells transfected with CFP plasmid to identify transfected cells.

Parmar V.K., Grinde E., Mazurkiewicz J.E, & Herrick-Davis K. (2016). Beta2-Adrenergic Receptor Homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression. Biochimica et biophysica acta, 1859(9 Pt A), 1445-1455.

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Culturing MCF-7 and HEK293 Cells

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MCF-7 cells (American Type Culture Collection (ATCC), Manassas, VA) were cultured in phenol-red free Eagle's Minimum Essential Medium (MEM, Corning cellgro, 17305-CV) supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 1% penicillin–streptomycin (Hyclone, SV30010), 1.5 g/L sodium bicarbonate (Corning cellgro, 25-035-CL), 1% l-glutamine (25-005-CI), and 10% fetal bovine serum (Corning cellgro, 35-010-CV). Human Embryonic Kidney 293 (HEK293) cells (ATCC) were cultured in phenol-red free Dulbecco's Modified Essential Medium (DMEM, Corning cellgro, 17-205-CV) supplemented with 1% l-glutamine, 1% penicillin–streptomycin, and 10% fetal bovine serum.

Smith L.C., Clark J.C., Bisesi JH J.r., Ferguson P.L, & Sabo-Attwood T. (2016). Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens,. Comparative biochemistry and physiology. Part D, Genomics & proteomics, 19, 159-173.

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Cytotoxicity Assay Using HEK293 Cells

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HEK293 cells (ATCC) were cultured in DMEM+10% FBS culture media in tissue culture treated (Corning, 430641U) flasks at 37 °C, 5% CO₂. The cells were plated at 5000 cells per well in 90 μL of respective media in tissue culture treated (Falcon, 353219) 96-well plates. 10 μL of test compound dilution prepared in culture media was added at different serially diluted concentrations (100 μM and lower) and incubated the plate for at least 72 hours at 37 °C. After the incubation period, 8 μL of Alamar blue dye (Invitrogen, DAL1100) was added to each well, the cells were incubated for 2–4 hours, and then analyzed using SpectraMax fluorescence reader using excitation and emission wavelengths of 544 nm and 590 nm, respectively. Data were expressed as mean ± standard error of mean for all experiments. The results were analyzed using a non-linear regression in Prism 7.0.1.

Shi D., Chahal K.K., Oto P., Nothias L.F., Debnath A., McKerrow J.H., Podust L.M, & Abagyan R. (2019). Identification of four amoebicidal non-toxic compounds by a molecular docking screen of Naegleria fowleri sterol Δ8−Δ7-isomerase and phenotypic assays. ACS infectious diseases, 5(12), 2029-2038.

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HEK-293 Cell Culture Protocol

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HEK-293 cells were purchased from American Type Culture Collection™ (CRL-1573™ Manassas, VA). HEK-293 cells were cultured in Minimum Essential Medium Eagle (MEM) with Earle’s salts and L-glutamine (Corning, Tewksbury, MA) with 10% FBS and maintained in 5% CO₂ atmosphere at 37°C. Routine subculture was performed as required depending on the degree of confluence.

Green B.L., Grant R.R., Richie C.T., Chatterjee B., De Melo M.S., Barr F.G., Pacak K., Agarwal S.K, & Nilubol N. (2022). Novel GLCCI1-BRAF fusion drives kinase signaling in a case of pheochromocytomatosis. European journal of endocrinology, 187(1), 185-196.

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In Vitro Transporter Assay Protocol

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Rifampin, diphenhydramide hydrochloride (98%), dimethyl sulfoxide (DMSO), ammonium acetate, acetonitrile Chromasolv (ACN), formic acid, dexamethasone, diclofenac, thiazolyl blue tetrazolium bromide (MTT), and methanol Chromasolv were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bosentan was purchased from Actelion Pharmaceuticals (Allschwil, Switzerland). Cryopreserved human hepatocytes, HEK293 cells overexpressing OATP1B1, OATP1B3, and OATP2B1 transporter, HEK293 mock cells, phosphate-buffered saline (PBS), Gentest High Viability CryoHepatocyte Recovery kit, hepatocyte culture medium, ITS+ culture supplement, Dulbecco’s Modification Eagle’s Medium (DMEM), Minimal Essential Medium (MEM) non-essential amino acid solution, epidermal growth factor (EGF), and Hank’s balanced salt solution (HBSS) were purchased from Corning (Corning, NY, USA). Penicillin/streptomycin, William’s E medium (without phenol red), fetal bovine serum (FBS), l-glutamine, gentamycin sulfate, SYBR Green ER qPCR Supermix, and Superscript First-strand Synthesis kit were purchased from Life Technologies (Burlington, ON, Canada). Sodium butyrate solution and testosterone were purchased from EMD-Millipore (Darmstadt, Germany) and Acros (Geel, Belgium), respectively. Easy Spin Total RNA extraction kit was purchased from iNtRON (Seongnam, Korea).

Han K.M., Ahn S.Y., Seo H., Yun J., Cha H.J., Shin J.S., Kim Y.H., Kim H., Park H.K, & Lee Y.M. (2017). Bosentan and Rifampin Interactions Modulate Influx Transporter and Cytochrome P450 Expression and Activities in Primary Human Hepatocytes. Biomolecules & Therapeutics, 25(3), 288-295.

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Dominant Negative FoxO1 in Insulin-Secreting Cells

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MIN6 and HEK293 cells were purchased from AddexBio Technologies and ATCC, respectively. MIN6 cells were maintained in DMEM with 4.5 g/L glucose and L-glutamine without sodium pyruvate (Corning) containing 15% fetal bovine serum (FBS), penicillin-streptomycin and 0.05 mM 2-mercaptoethanol. For gene expression analysis, MIN6 cells were transduced with adenovirus encoding dominant negative FoxO1 and GFP at an MOI of 500 and incubated for 48 h. Cells were also treated with FoxO1 inhibitor (AS18420856, 100 nM) and incubated for 24 h. HEK293 cells were maintained in DMEM with 1.0 g/L glucose and L-glutamine without sodium pyruvate (Corning) containing 10% FBS and penicillin-streptomycin. Islets were isolated by collagenase digestion as previously described [17 (link)]. Isolated islets were incubated overnight in RPMI 1640 containing 10% FBS with penicillin-streptomycin and used for further analyses.

Miyachi Y., Kuo T., Son J, & Accili D. (2021). Aldo-ketoreductase 1c19 ablation does not affect insulin secretion in murine islets. PLoS ONE, 16(11), e0260526.

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Culturing HEK293 and 293-Cre Cells

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HEK293 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and 293-Cre cells were provided as a kind gift from Dr. Frank Graham [37 (link)]. HEK293 cells were cultured in standard growth medium containing high-glucose DMEM (Corning, New York, NY, USA), 10% FB Essence (VWR Life Science Seradigm, Radnor, PA, USA), and 1% Pen-Strep (Corning). The standard growth medium used to maintain 293-Cre cells was similar to what was used for the HEK293 cells, with the only exception being the addition of G418 (500 μg/mL; VWR Life Science Seradigm) to the standard growth medium used for culturing 293-Cre cells. Medium for both cell types was replenished every 48–72 h. For cell maintenance, both HEK293 cells and 293-Cre cells were incubated within 10 cm cell culture plates at 37 °C under 5% CO₂ conditions, and the confluency of cultured cells was based on coverage [40 ].

Esobi I., Olanrewaju O., Echesabal-Chen J, & Stamatikos A. (2022). Utilizing the LoxP-Stop-LoxP System to Control Transgenic ABC-Transporter Expression In Vitro. Biomolecules, 12(5), 679.

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Cell Culture and Organ Culture Protocols

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HEK293 cells (female) and Neuro2a cells (male) were obtained from
the American Type Culture Collection (ATCC). HEK293 cells were grown in
Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12
from Corning). Neuro2a cells were grown in DMEM (Corning). Both DMEM/F-12
and DMEM were supplemented with 10% fetal bovine serum (FBS from Atlanta
Biologicals), penicillin (100 units/mL; Thermo Fisher Scientific), and
streptomycin (100 μg/mL; Thermo Fisher Scientific). Organs of Corti
were dissected from P0–P5 mice and placed on Matrigel-coated
transwell plates (Corning). The sexes of these mice were not determined.
Organ cultures were maintained in neurobasal-A medium supplemented with
B-27, N-2, 0.5 mM L-glutamine (all from Thermo Fisher Scientific), and 100
U/mL penicillin (Sigma). The culture medium was replaced on the organs once
in every 3 days. Organ cultures and cell lines were grown at 37 °C in
the presence of 5% CO₂.

Nakano Y., Kelly M.C., Rehman A.U., Boger E.T., Morell R.J., Kelley M.W., Friedman T.B, & Bánfi B. (2018). Defects in the alternative splicing-dependent regulation of REST cause deafness. Cell, 174(3), 536-548.e21.

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Maintenance and Transfection of Cell Lines

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HEK293 cells, NIH3T3 cells and HepG2 cells were purchased from ATTC. Authentication of the cell lines was performed by ATCC according to their protocol. Cultures were tested for mycoplasma contamination and all cell lines used for experiments were mycoplasma-free. HEK293 cells and NIH3T3 cells were cultured in DMEM (Corning Inc.) containing 10% FBS and 100 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific) and maintained by standard methods. HepG2 cells were maintained in MEM containing 10% FBS, non-essential amino acids and 1 mM Sodium Pyruvate. Mouse embryonic fibroblasts (MEFs) were prepared from E12.5 embryos of Per2^luc knockin mice (Yoo et al., 2004 (link)). After removing the head, paws and internal organs, embryos were chopped and incubated in 0.25% trypsin in PBS for 24 hr at 4°C. After incubation for 30 min at 37°C in 0.25% trypsin in PBS, cells were dissociated by pipetting in DMEM. Supernatant was cultured in a cell culture dish with DMEM and maintained by standard methods. Cells were transfected with Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) according to manufacturer’s protocol. DNA constructs used for transfections are listed in supplemental experimental procedures (Hirano et al., 2016b (link)).

Hirano A., Braas D., Fu Y.H, & Ptáček L.J. (2017). FAD regulates CRYPTOCHROME protein stability and circadian clock in mice. Cell reports, 19(2), 255-266.

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ACE2-Mediated SARS-CoV-2 Pseudovirus Entry Inhibition

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HEK 293 cells (ATCC^® CRL-1573) stably over-expressing full-length human ACE2 protein were seeded in 96-well white polystyrene microplates (Corning, Cat. No. CLS3610) at 0.03 × 10⁶ cells/well in DMEM (10% FBS and 1% Pen-Strep), and grown overnight at 37 °C, 5% CO₂. To test the inhibition of pseudovirus entry by ACE2 WT or mutants, increasing concentration of Fc-tagged ACE2 proteins were first mixed with pseudoviruses and incubated at room temperature for 10 m⁷². The ACE2 WT or mutant treated pseudovirus mixture was then used to infect cells. The cells were incubated at 37 °C, 5% CO₂ for 6 h, then the medium was replaced with fresh DMEM (10% FBS and 1% Pen-Strep), and then again every 24 h for up to 72 h. To measure the luciferase signal (a proxy for virus uptake), DMEM was removed and cells were replaced in DPBS (ThermoFisher, Cat. No. 14190250) and mixed with an equal volume of ONE-Glo™ EX Luciferase Assay System (Promega, Cat. No. 8130). Relative luciferase units were measured using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data was then analyzed using GraphPad Prism Version 8.4.3 (GraphPad Software, LLC.).

Suryamohan K., Diwanji D., Stawiski E.W., Gupta R., Miersch S., Liu J., Chen C., Jiang Y.P., Fellouse F.A., Sathirapongsasuti J.F., Albers P.K., Deepak T., Saberianfar R., Ratan A., Washburn G., Mis M., Santhosh D., Somasekar S., Hiranjith G.H., Vargas D., Mohan S., Phalke S., Kuriakose B., Antony A., Ustav Jr M., Schuster S.C., Sidhu S., Junutula J.R., Jura N, & Seshagiri S. (2021). Human ACE2 receptor polymorphisms and altered susceptibility to SARS-CoV-2. Communications Biology, 4, 475.

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