Hek293

Manufactured by Thermo Fisher Scientific

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The HEK293 is a widely used human embryonic kidney cell line. It is a well-established and characterized cell line commonly used in laboratory research and experiments.

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HEK293-EBNA Flp-In T-REx HEK293 cell line HEK293 Flp-In T-REx HEK293 (293-H) cells Flp-In HEK293 HEK293-6E HEK293-FT packaging cells HEK293 cell line HEK293 Flp-In T-Rex cells Hek293 EBNA cells

267 protocols using hek293

Validating hcmv-miR-UL59 Targeting ULBP1

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The human embryonic kidney cell line HEK293 was obtained from the Shanghai Institutes for Biological Sciences (Shanghai, China). The complete growth medium of HEK293 was Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, NY, USA) and 10% foetal bovine serum (Gibco, NY, USA). These cells were maintained at 37 °C under an atmosphere of 5% CO₂—95% air. Luciferase reporter assays were performed to confirm that hcmv-miR-UL59 directly targeted the 3′UTR of the ULBP1 gene. The 3′UTR fragments from ULBP1 containing the predicted hcmv-miR-UL59 binding site were cloned into a pMIR-reporter plasmid (Ambion, Shanghai, China), and which was co-transfected into HEK293 cells with hcmv-miR-UL59 mimics by Lipofectamine 2000 (Thermo fisher, NY, USA). A β-galactosidase vector was co-transfected into HEK293 cells simultaneously as a transfection control. Cells were assayed using luciferase assay kits (Promega, Madison, WI, USA) at 24 h after transfection. The reported data represent three independent experiments.

Ding M., Wang X., Wang C., Liu X., Zen K., Wang W., Zhang C.Y, & Zhang C. (2017). Distinct expression profile of HCMV encoded miRNAs in plasma from oral lichen planus patients. Journal of Translational Medicine, 15, 133.

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CRISPR Genome Editing Experiments

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Cell culture experiments were performed on human U2OS (gift from T. Cathomen), HEK293 (Thermo-Fisher), K562, and PGP1 fibroblast cells (gift from G. Church). U2OS and HEK293 cells were cultured in Advanced DMEM (Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax (Life Technologies) and penicillin/streptomycin at 37°C with 5% CO₂. K562 cells were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax and penicillin/streptomycin at 37°C with 5% CO₂. Human PGP1 fibroblasts were cultured in Eagle’s DMEM (ATCC) with 10% FBS, 2 mM GlutaMax and penicillin/streptomycin at 37°C with 5% CO₂. For CIRCLE-seq experiments, genomic DNA was isolated using Gentra Puregene Tissue Kit (Qiagen) and quantified by Qubit (Thermo Fisher). For targeted tag-integration deep-sequencing experiments, U2OS cells (program DN-100), HEK293 cells (program CM-137), and K562 cells (program FF-120) were transfected in 20 µl Solution SE (Lonza) on a Lonza Nucleofector 4-D, according to the manufacturer’s instructions. In U2OS cells, 500 ng of pCAG-Cas9 (pSQT817), 250 ng of gRNA encoding plasmids, and 100 pmol of GUIDE-seq end-protected dsODN were cotransfected. Genomic DNA for targeted tag integration sequencing was harvested approximately 72 hours post transfection using the Agencourt DNAdvanced Genomic DNA Isolation Kit (Beckman Coulter Genomics).

Tsai S.Q., Nguyen N.T., Malagon-Lopez J., Topkar V.V., Aryee M.J, & Joung J.K. (2017). CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nature methods, 14(6), 607-614.

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HEK293 Cell Transfection and Lysis

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Human embryonic kidney 293FT cells (HEK293; ThermoFisher) were transfected in 25 cm² flat bottom flasks with each relevant DNA construct in 500 μL serum-free Dulbecco's modified Eagle's medium (DMEM) using PolyJet reagent (SignaGen Laboratories Rockville, MD) in a 3 : 1 (reagent volume : DNA mass) ratio. Empty vector DNAs were used to transfect equal DNA concentrations. Cells were incubated overnight, medium was aspirated from each flask, and cells were washed with cold phosphate-buffered saline (PBS). HEK293 cells were then suspended in a low ionic Tris homogenization buffer (0.01 M DTT, 0.005 M EDTA, 0.002 M Tris-HCl pH 7.5, 1% Triton X-100) containing protease inhibitors (HALT; ThermoFisher) and phosphatase inhibitors (20 mM sodium fluoride, 20 mM sodium orthovanadate, 20 mM β-glycerophosphate, and 10 mM sodium pyrophosphate; Sigma-Aldrich, St. Louis, MO or ThermoFisher).

Hiday A.C., Edler M.C., Salek A.B., Morris C.W., Thang M., Rentz T.J., Rose K.L., Jones L.M, & Baucum AJ I.I. (2017). Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction. Neural Plasticity, 2017, 4153076.

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HEK293 Cell Culture and Receptor Expression

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HEK293 (WT) were obtained from the American Type Culture Collection (ATCC), parental HEK293 (WT) and Flp-In T-REx293 were from ThermoFisher. HEK293 (WT), parental and CRISPR/Cas9 genome-edited HEK293 cells were cultured in DMEM (ThermoFisher) containing 10% fetal bovine serum (FBS, PAN biotech), 100 U ml⁻¹ Penicillin, 100 mg ml⁻¹ Streptomycin (ThermoFisher) at 37 °C and 5% CO₂. Receptor expressing HEK293 cell lines were generated by stable transfection of receptor cDNA (GPR17, DP2, β2AR, AT1R, and M3-DREADD cloned into pcDNA3.1(+)) using Fugene HD (Promega) according to manufacturer's instructions and subsequently cultivated using growth medium containing 500 µg ml⁻¹ G418 (Invivogen). FFA2 receptor was expressed in Flp-In T-REx293 cells upon induction of expression after treatment with 1 µg ml⁻¹ doxycycline for at least 16 h. Transiently transfected cells were analyzed 24–48 h after transfection using Fugene HD (Promega). All cell lines were checked for and free of mycoplasma contamination. All chemicals were purchased from Sigma-Aldrich unless otherwise indicated. For G protein inhibition, cells were incubated with pertussis toxin (PTX) for at least 18 h at 150 ng ml⁻¹ and FR for at least 1 h at 1 µM.

Grundmann M., Merten N., Malfacini D., Inoue A., Preis P., Simon K., Rüttiger N., Ziegler N., Benkel T., Schmitt N.K., Ishida S., Müller I., Reher R., Kawakami K., Inoue A., Rick U., Kühl T., Imhof D., Aoki J., König G.M., Hoffmann C., Gomeza J., Wess J, & Kostenis E. (2018). Lack of beta-arrestin signaling in the absence of active G proteins. Nature Communications, 9, 341.

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CRISPR Genome Editing Experiments

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HEK293 Cell Transfection and Lysis

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Human embryonic kidney 293FT cells (HEK293; ThermoFisher) were transfected in 25 cm² flat bottom flasks with each relevant DNA construct in 500 μL serum-free Dulbecco’s modified Eagle’s medium using PolyJet reagent (SignaGen Laboratories Rockville, MD) in a 3:1 (reagent volume:DNA mass) ratio. Empty vector DNAs were used to transfect equal DNA concentrations. Cells were incubated overnight, medium was aspirated from each flask, and cells were washed one time with cold phosphate-buffered saline (PBS). HEK293 cells were then suspended in a KCl homogenization buffer (150 mM KCl, 50 mM Tris-HCl pH 7.5, 1 mM DTT, 2 mM EDTA, 1% Triton X-100) or RIPA homogenization buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% NP-40, 1% Deoxycholate) containing protease inhibitors (ThermoFisher or Bimake, Houston, TX) and phosphatase inhibitors (20 mM sodium fluoride, 20 mM sodium orthovanadate, 20 mM β-glycerophosphate, and 10 mM sodium pyrophosphate; MilliporeSigma, St. Louis, MO or ThermoFisher).

Morris C.W., Watkins D.S., Salek A.B., Edler M.C, & Baucum AJ I.I. (2018). The association of spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5.. Molecular and cellular neurosciences, 90, 60-69.

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Optimizing GARP Protein Expression in HEK293 Cells

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Human embryonic kidney cells (HEK293) were obtained from American Type Culture Collection (No. CRL-1573). HEK293 cells were transfected with plasmid DNA using Lipofectamine^TM LTX (Life Technologies Invitrogen) according to the manufacturer’s instructions. Briefly, 0.25 × 10⁶ cells were seeded in 12-well plates and transfected on the following day with 0.4 μg pcDNA3.1-GARPcds variants GARP_wt, GARP_c.741G>A, GARP_c.934C>T, and GARP_c.1262G>A (Thermo Fisher Scientific) or the empty pcDNA3.1 vector as a control. Plasmid DNA was dissolved in 200 µl Opti-MEM and incubated with 3.5 µl Lipofectamine LTX (both from Life Technologies Invitrogen) for 30 min at room temperature. The DNA/Lipofectamine complexes were added to HEK293 cells cultured in 1 ml Dulbecco’s modified Eagle’s medium supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM l-glutamine, and 10% FCS (all from Life Technologies Invitrogen). The expression of GARP was analyzed 48 h after transfection by flow cytometry.

Lehmkuhl P., Gentz M., Garcia de Otezya A.C., Grimbacher B., Schulze-Koops H, & Skapenko A. (2021). Dysregulated immunity in PID patients with low GARP expression on Tregs due to mutations in LRRC32. Cellular and Molecular Immunology, 18(7), 1677-1691.

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HEK-293 Cell Line Transfection Protocol

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The human embryonic kidney cell line HEK-293 (ATCC Certified from LGC Standards, Milan, Italy) was grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS).
5 × 10⁶ HEK-293 cells, cultured overnight in 100 mm tissue culture dishes, were stably transfected with 10 μg of pPLR2.5, or pcDNA3 and 60 μl of LipofectAMINE (Invitrogen) for 5 h at 37°C (5% CO2). Transfected cells, named LR-293 and V-293 respectively, were selected by Geneticin (GIBCO) at 1.5 mg/ml, pooled, and cultured in the presence of 0.5 mg/ml Geneticin.
67LR highly expressing HT1080 fibrosarcoma [30 (link)] and MDAMB231 breast cancer cell lines [43 (link)] were grown in DMEM supplemented with 10% FBS.

Pesapane A., Di Giovanni C., Rossi F.W., Alfano D., Formisano L., Ragno P., Selleri C., Montuori N, & Lavecchia A. (2015). Discovery of new small molecules inhibiting 67 kDa laminin receptor interaction with laminin and cancer cell invasion. Oncotarget, 6(20), 18116-18133.

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Stable Overexpression of Human TRP Channels in HEK-293 Cells

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Human embryonic kidney-293 (HEK-293) cells (ATCC, Rockville, MD) were engineered to stably overexpress human TRPV3 (in pcDNA3.1 D V5/His-TOPO) and other human TRP channels (TRPA1, TRPM8, TRPV1, TRPV2, and TRPV4) as previously described (Deering-Rice et al. 2011 (link)). HEK-293 cells were grown in DMEM:F12 (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (FBS) and 1× penicillin/streptomycin (Invitrogen). HEK-293 cells stably overexpressing the various TRP channels were maintained in DMEM:F12 supplemented with 5% FBS, 1× penicillin/streptomycin, and 350 μg/mL Geneticin (Invitrogen). Cells were subcultured using trypsin and plated into 1% gelatin-coated 96- or 384-well plates for calcium imaging experiments. Human immortalized keratinocytes, HaCaT cells, were provided by Dr. Douglas Grossman, M.D., Ph.D., of the Department of Dermatology, University of Utah. HaCaT cells were cultured in DMEM containing 5% FBS and 1× penicillin/streptomycin/glutamine. Cells were subcultured using trypsin and plated into 96-well plates for experiments.

Deering-Rice C.E., Mitchell V.K., Romero E.G., Abdel Aziz M.H., Ryskamp D.A., Križaj D., Venkat R.G, & Reilly C.A. (2014). Drofenine: a 2-APB analog with improved selectivity for human TRPV3. Pharmacology Research & Perspectives, 2(5), e00062.

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Stable Cell Lines Expressing HA-tagged HPV-E6

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TERT-immortalized human epidermal keratinocytes N/TERT-1 cell line were obtained from Dr. James Rheinwald (Dept. of Dermatology, Harvard Skin Disease Research Center). This cell line was then used to generate stable HA tagged HPV-E6 expressing cell lines. N/TERT-1 cells and generated N/TERT-1 cells expressing HA tagged E6 cell lines were maintained in EpiLife medium (Life technologies) containing human keratinocyte growth supplement (HKGs) and penicillin-streptomycin (100U/ml). HEK293 (American Type Culture Collection) was also used to generate stable HA tagged HPV-E6 expressing cell lines. HEK293 cells and generated HEK293 cells expressing HA tagged E6 cell lines were maintained in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum and penicillin-streptomycin.

Wang J., Dupuis C., Tyring S.K, & Underbrink M.P. (2016). Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins. PLoS ONE, 11(2), e0149859.

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