Hek293a cells

Manufactured by American Type Culture Collection

Sourced in United States

HEK293A cells are a widely used human embryonic kidney cell line derived from human embryonic kidney cells. They are commonly used in cell biology research, protein expression, and virus production. The cells exhibit a stable epithelial-like morphology and can be easily transfected with a variety of expression vectors.

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HEK293A (53 citations) HEK293A cell line (5 citations)

24 protocols using hek293a cells

Generation and Characterization of Adenoviral Constructs

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The generation of GFP-encoding adenovirus (Ad-GFP) was described previously^{72 (link)}. HSF1-FLAG adenovirus (Ad-HSF1) and HSF1-AB-FLAG adenovirus (Ad-HSF1-AB) were made using ViraPower adenoviral expression system (Thermo Fisher Scientific) following manufacturer’s instruction. HSF1-Flag was cloned from a pCMV-HSF1-Flag plasmid (Addgene, Cambridge, MA; plasmid number 1932), and HSF1-AB-Flag was amplified from pCMV-AB-Flag^{15 (link)}. The primers used for both constructs are as follows: Ad-HSF1 Forward, 5-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGA TCTGCCCGTGGGCCC-3, and Ad-HSF1 Reverse, 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTGTTATTACTTATCGTCGTCATCCTTGTAATC-3. The PCR products were cloned into a shuttle vector pDONR221 and then recombined with a destination vector pAd/CMV/V5-DEST. The adenovirus was amplified in the HEK293A cells (ATCC, Manassas, VA) and subsequently purified using CsCl density gradient centrifugation followed by dialysis with Phosphate-Buffered Saline (PBS). The multiplicity of infection for neuronal cultures was approximately 10.

Qu Z., Titus A.S., Xuan Z, & D’Mello S.R. (2018). Neuroprotection by Heat Shock Factor-1 (HSF1) and Trimerization-Deficient Mutant Identifies Novel Alterations in Gene Expression. Scientific Reports, 8, 17255.

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HEK 293A and AR42J Cell Transfection Protocol

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HEK 293A cells were purchased from ATCC (Manassas, VA) and maintained in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin (#15140-122 GIBCO, Life Technologies, Grand Island, NY) at 37 °C in a 5% CO₂-humidified incubator. Sixteen hours prior to transfection, cells (2 ml/ well for 6-well plates and 10 ml/dish for 10-cm culture dishes) were seeded at 75% confluence. Transfections were carried out using FuGENE 6 (Roche Diagnostics, Indianapolis, IN). Briefly, in 100 μl of Opti-MEM I reduced serum medium (Invitrogen), 5 μl of FuGENE 6 was mixed with 1.65 μg of plasmid DNA (pcDNA3, pcDNA3/PNLIP, or pcDNA3/ PNLIP p.T221M). The mixture was then added to the cells in each well of 6-well plates. For RNA extraction, a mixture of 25 μl of FuGENE 6 and 10 μg of each plasmid DNA in 500 μl of Opti-MEM I reduced serum medium was added for each 10-cm dish.
AR42J rat pancreatic acinar cells (ATCC #CRL-1492) were maintained in DMEM supplemented with 20% fetal bovine serum, 4 mM glutamine and 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C. Prior to transfection, cells were plated in 6-well plates at a density of 10⁶ cells per well and were grown in the presence of 100 nM dexamethasone for 48 h. Transduction with adenovirus were performed in 1 ml OptiMEM containing 100 nM dexamethasone using the indicated viral concentrations.

Szabó A., Xiao X., Haughney M., Spector A., Sahin-Tóth M, & Lowe M.E. (2015). A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding. Biochimica et biophysica acta, 1852(7), 1372-1379.

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Culturing A549, A549-ΔMAVS, and HEK 293 A Cells

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A549 [American Type Culture Collection (ATCC), Manassas, VA, USA], A549-ΔMAVS cells (48 (link)), and the human embryonic kidney (HEK) 293 A cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with heat-inactivated 10% fetal bovine serum (FBS) and 2 mM L-glutamine (all purchased from Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in 5% CO₂ atmosphere.

Bougon J., Kadijk E., Gallot-Lavallee L., Curtis B.A., Landers M., Archibald J.M, & Khaperskyy D.A. (2024). Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells. Journal of Virology, 98(5), e01901-23.

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Cultivation of Diverse 293-Derived Cell Lines

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The 293SF-3F6²⁷ (link) and the 293SF-CymR cells were cultured in SFM4-Transfx-293 medium (Hyclone, South Logan, UT) supplemented with 6 mM L-glutamine (Hyclone). 293SF-CymR/λR-GyrB cells were generated using low-calcium-serum-free medium (LC-SFM) (Gibco, Life Technology Corporation, Grand Island, NY), supplemented with 6 mM L-glutamine and 10 mg/mL rTransferin (Biogems, Westlake Village, CA) and expanded in suspension culture in SFM4-Transfx-293. The packaging cells (293SF-PacLVIIIb) and producer clones were generated using a mixture of 50% LC-SFM supplemented with 6 mM L-glutamine and 10 mg/mL rTransferin and 50% HyCell-Transfx-H (Hyclone) supplemented with 4 mM L-glutamine and 0.1% kolliphor (Sigma-Aldrich, St. Louis, MO). They were maintained in suspension culture in 100% HyCell-Tranfx-H. For suspension culture, the cells were grown in shake flasks (Corning, Oneonta, NY) at 120 rpm with an orbital diameter of 25 mm. The HEK293A cells (ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) supplemented with 5% fetal bovine serum (FBS; Hyclone) and 2 mM L-glutamine using tissue culture-treated dishes. All the cell lines were maintained at 37°C in a 5% CO₂ humidified atmosphere.

Broussau S., Lytvyn V., Simoneau M., Guilbault C., Leclerc M., Nazemi-Moghaddam N., Coulombe N., Elahi S.M., McComb S, & Gilbert R. (2023). Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning. Molecular Therapy. Methods & Clinical Development, 29, 40-57.

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PAMAM Dendrimer Purification and Characterization

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Biomedical grade G5 PAMAM dendrimer was purchased from Dendritech Inc. and purified using rp-HPLC to give a molecular weight fraction free of trailing generations (G1–G4) as well as G5 dimers and higher oligomers.^{28 (link)} Trifluoroacetic acid, HPLC grade water, GE PD-10 Sephadex columns, and HPLC grade acetonitrile were purchased from Fisher-Scientific and used as received. 5-carboxy tetramethylrhodamine succinimydyl ester (TAMRA) was purchased from Life Technologies. A 500 MHz Varian NMR instrument was used for all ¹H and ¹⁹F NMR measurements. All MALDI-TOF MS measurements were performed on a Bruker Ultraflex III with sinapinic acid matrix (Sigma Aldrich) and sodium trifluoroacetate (Fischer Scientific) salt sample preparation. Serum-free DMEM (SFM) from life technologies was employed for cell culture of HEK293A cells, which were obtained from ATCC. Complete medium was made by adding 50 mL of fetal bovine serum (FBS) and 5 mL 100× of penicillin streptomycin to 500 mL of SFM.

Dougherty C.A., Vaidyanathan S., Orr B.G, & Banaszak Holl M.M. (2015). Fluorophore:Dendrimer Ratio Impacts Cellular Uptake and Intracellular Fluorescence Lifetime. Bioconjugate chemistry, 26(2), 304-315.

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Cell Culture Protocols for HEK293A and 3T3-L1

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HEK293A cells (ATCC) were cultured at 37 °C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Invitrogen, USA), 1% penicillin and streptomycin (P/S, P4333, Sigma-Aldrich). 3T3-L1 preadipocytes (ATCC) were cultured at 37 °C in 5% CO₂ in high glucose DMEM containing 10% new calf serum (NCS, Invitrogen) and 1% P/S. The medium for both cell lines was refreshed every second day, with transfer accomplished by 10% trypsin digest when cells were at ~ 70% confluence.

Shin K., Landsman M., Pelletier S., Alamri B.N., Anini Y, & Rainey J.K. (2018). Proapelin is processed extracellularly in a cell line-dependent manner with clear modulation by proprotein convertases. Amino Acids, 51(3), 395-405.

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Cell Culture Conditions for HEK293A and hTERT A41hBAT-SVF

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HEK293A cells (ATCC) were cultured in DMEM medium with 10% cosmic calf serum (CCS) and 1% penicillin-streptomycin. The hTERT A41hBAT-SVF was grown in BGM medium.

Sebag S.C., Zhang Z., Qian Q., Li M., Zhu Z., Harata M., Li W., Zingman L.V., Liu L., Lira V.A., Potthoff M.J., Bartelt A, & Yang L. (2021). ADH5-mediated NO bioactivity maintains metabolic homeostasis in brown adipose tissue. Cell reports, 37(7), 110003.

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Generating Rat mGlu2 Stable Cell Line for Calcium Assay

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In order to generate a rat mGlu₂ stable cell line to
be used for a calcium mobilization assay, HEK293A cells (ATCC) were
transfected with mouse G_α15-pCMV6 plasmid (Origene)
using Fugene6 (Promega). The cells were selected for G_α15 expression with 1 mg/mL G418. Two weeks after the selection, polyclonal
HEK293A G_α15 cells were obtained. The entire coding
sequence of rat mGlu₂ was amplified by PCR and cloned into
the expression plasmid pIRESpuro3 (Invitrogen). Rat mGlu₂-pIRESpuro3 was transfected into HEK293A G_α15 cells
and selected for mGlu₂ expression with 0.6 μg/mL
puromycin in the presence of G418. The resulting polyclonal HEK293A
mGlu₂ G_α15 cells were then utilized for
calcium mobilization assays. Cells were maintained in growth medium
containing DMEM, 10% FBS, 20 mM HEPES, 2 mM l-glutamine,
antibiotic/antimycotic, nonessential amino acids, 700 μg/mL
G418, and 0.6 μg/mL puromycin at 37 °C in the presence
of 5% CO₂.

Dhanya R.P., Sheffler D.J., Dahl R., Davis M., Lee P.S., Yang L., Nickols H.H., Cho H.P., Smith L.H., D’Souza M.S., Conn P.J., Der-Avakian A., Markou A, & Cosford N.D. (2014). Design and Synthesis of Systemically Active Metabotropic Glutamate Subtype-2 and -3 (mGlu2/3) Receptor Positive Allosteric Modulators (PAMs): Pharmacological Characterization and Assessment in a Rat Model of Cocaine Dependence. Journal of Medicinal Chemistry, 57(10), 4154-4172.

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Propagation and Purification of HP-PRRSV SD-JN

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HP-PRRSV strain SD-JN was kept in our laboratory [27] (link). MARC-145 cells were used to propagate and titrate HP-PRRSV SD-JN strain. The infected cell lysates were clarified, titrated, diluted to 1×10⁵ TCID₅₀/ml and stored at −20°C to be used for animal challenge. The sixth passage (F6) MARC-145 cell culture supernatant was filtrated through a 0.45 µm filter and SD-JN PRRSV was concentrated from the supernatant by ultracentrifugation at 120,000×g (SW 40 rotor, Beckman) at 4°C for 2 h. The virus pellet was collected, diluted with TNE buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA] and then layered on the top of 25–65% (w/v) sucrose gradients and at the same time centrifuged at 120,000×g (SW 40 rotor, Beckman) at 4°C for 4 h. The PRRSV particles band were harvested and pelleted at 120,000×g at 4°C for 2 h to remove the traces of sucrose. Then the PRRSV pellet was resuspended in phosphate buffered saline (PBS), quantitated by optical density (OD) measurement as described previously [6] (link) and used for indirect ELISA (iELISA), T lymphocyte proliferation and cytokine assays. HEK-293A cells (ATCC CRL1573) were used for transfection of plasmids. All cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U penicillin/ml and 100 µg streptomycin/ml.

Du Y., Lu Y., Wang X., Qi J., Liu J., Hu Y., Li F., Wu J., Guo L., Liu J., Tao H., Sun W., Chen L., Cong X., Ren S., Shi J., Li J., Wang J., Huang B, & Wan R. (2014). Highly Efficient Expression of Interleukin-2 under the Control of Rabbit β-Globin Intron II Gene Enhances Protective Immune Responses of Porcine Reproductive and Respiratory Syndrome (PRRS) DNA Vaccine in Pigs. PLoS ONE, 9(3), e90326.

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Cell Culture Conditions for HEK293A and hTERT A41hBAT-SVF

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HEK293A cells (ATCC) were cultured in DMEM medium with 10% cosmic calf serum (CCS) and 1% penicillin-streptomycin. The hTERT A41hBAT-SVF was grown in BGM medium.

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