Human hek293t cells

Manufactured by American Type Culture Collection

Sourced in United States

Human HEK293T cells are a widely used cell line derived from human embryonic kidney cells. They are transformed with the adenovirus 5 DNA and express the SV40 large T antigen. HEK293T cells are commonly used as a host for the production of recombinant proteins and the study of gene expression and regulation.

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28 protocols using human hek293t cells

Cell Culture and Transfection

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Human HEK293T cells (female) were obtained from ATCC (Virginia, USA) and the human GloResponse™ NFAT-RE-luc2P HEK293 cell line (female) was obtained from Promega Corporation (Wisconsin, USA). Human umbilical endothelial (HUVEC) cells (newborn male, single donor) were obtained from Thermo Fisher Scientific (Waltham, USA). HUVECs and HEK293T cells were transfected and cultured as described in Method Details.

Kilpatrick L.E., Alcobia D.C., White C.W., Peach C.J., Glenn J.R., Zimmerman K., Kondrashov A., Pfleger K.D., Ohana R.F., Robers M.B., Wood K.V., Sloan E.K., Woolard J, & Hill S.J. (2019). Complex Formation between VEGFR2 and the β2-Adrenoceptor. Cell Chemical Biology, 26(6), 830-841.e9.

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Culturing Drosophila and Mammalian Cells for Protein Expression

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Drosophila S2 cells (Gibco, NY, USA, R69007) were maintained in SFX-insect medium (HyClone, Logan, UT, SH30278.02) at 28°C. Human HEK 293T cells (ATCC, Manassas, USA, CRL-3216) and SRSF2 protein-depleted SRSF2-MEFs were maintained in DMEM (GIBCO, NY, USA, C11965500BT) supplemented with 10% FBS (BI, Beit-Haemek, Israel, 04-001-1A) or tet-free FBS (Clontech, CA, USA, 631106) under humidified 5% CO2/95% air at 37°C. SRSF2-MEFs were treated with 10 μg/mL doxycycline (DOX, Sigma, MO, USA, D9891-1G) for 1 day to deplete SRSF2 transcription (DOX+) [48 (link)]. We used HEK 293T cells to express the locust SRSF2-V5 fusion protein because this cell line represents a rapid and high-yield protein production system and express more locust SRSF2 than Drosophila S2 cells. Lastly, we adopted the SRSF2-MEF cell line to verify the interaction of SRSF2 with PAHAL by using an endogenous SRSF2-KO system constructed in the cell line.

Zhang X., Xu Y., Chen B, & Kang L. (2020). Long noncoding RNA PAHAL modulates locust behavioural plasticity through the feedback regulation of dopamine biosynthesis. PLoS Genetics, 16(4), e1008771.

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Diverse Cell Lines for COVID-19 Research

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Saccharomyces cerevisiae YVH10 cells (ATCC) were used in antibody library generation and FACS sort. Hela-hACE2 cells (Rogers et al., 2020 (link)) were used in pseudovirus neutralization assay. Saccharomyces cerevisiae EBY100 cells (ATCC) were used in RBD mutant library generation and FACS sort. Human HEK293T cells (ATCC) were used for pseudovirus production. FreeStyle HEK293 cells (ThermoFisher) were used for recombinant S protein production. Expi293F cells (ThermoFisher) were used for monoclonal antibody and recombinant RBD production. Vero-E6 cells (ATCC) were used for live virus plaque assay.

Zhao F., Keating C., Ozorowski G., Shaabani N., Francino-Urdaniz I.M., Barman S., Limbo O., Burns A., Zhou P., Ricciardi M.J., Woehl J., Tran Q., Turner H.L., Peng L., Huang D., Nemazee D., Andrabi R., Sok D., Teijaro J.R., Whitehead T.A., Ward A.B., Burton D.R, & Jardine J.G. (2022). Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape pathways. iScience, 25(9), 104914.

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Luciferase Reporting of CrPV-Fluc mRNA

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Polyadenylated CrPV-Fluc mRNA was in vitro transcribed using a 50T-tailed PCR product as a template, as described previously (Prokhorova et al. 2016 (link)). Human HEK293T cells (ATCC) were seeded in DMEM supplemented with 10% FBS onto a 96-well white opaque FB plate (Greiner). 12–16 h later, the cells were transfected with 30 ng of mRNA per well using GenJector-U (Molecta, Russia). Beetle luciferin (Promega) was added to the medium to final concentration of 0.5 mM, and the plate was immediately placed into Infinite 200 Pro microplate reader (TECAN) provided with a gas control module. The cells were incubated at 36.6°С in 5% CO₂ for 12 h, luciferase activity was continuously measured every 5 min with integration time 5 s, as describes earlier (Panova et al. 2023 (link)).

Lidsky P.V., Yuan J., Lashkevich K.A., Dmitriev S.E, & Andino R. (2023). Monitoring integrated stress response in live Drosophila. bioRxiv.

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Cell Line Authentication and Validation

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NIH3T3 and MEF cells were obtained from ATCC in 2012 and 2015, respectively and used within three months of receipt. Human T-ALL cell lines, MOLT-4, CCRF-CEM, HPB-ALL, JURKAT, DND-41, KE-37, KOPTK1, MOLT13, P12-ICHIKAWA, PEER were a gift from A. Thomas Look in 2012. Human HEK293T cells were ordered from ATCC in 2012. All human cell lines were authenticated at receipt and just prior to use in experiments using Small Tandem Repeat profiling and certified mycoplasma (MycoAlert Plus, Lonza, tested every 6 months).

Lobbardi R., Pinder J., Martinez-Pastor B., Theodorou M., Blackburn J.S., Abraham B.J., Namiki Y., Mansour M., Abdelfattah N.S., Molodtsov A., Alexe G., Toiber D., de Waard M., Jain E., Boukhali M., Lion M., Bhere D., Shah K., Gutierrez A., Stegmaier K., Silverman L.B., Sadreyev R.I., Asara J.M., Oettinger M.A., Haas W., Look A.T., Young R.A., Mostoslavsky R., Dellaire G, & Langenau D.M. (2017). TOX regulates growth, DNA repair, and genomic instability in T-cell Acute Lymphoblastic Leukemia. Cancer discovery, 7(11), 1336-1353.

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Establishing GABAAR Expressing Cell Lines

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Human HEK293T cells (ATCC) and mouse GT1-7 cells (Professor Pamela Mellon, UCSD) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone) with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% Penicillin-Streptomycin (Hyclone) at 37 °C in 5% CO₂. Cells were transfected using TransIT-2020 (Mirus). The HEK293T cell line that stably expressing α1β2γ2 GABA_ARs was generated by transient transfection with α1:β2:γ2 (1:1:1) plasmids and selected using G-418. For siRNA transfections, cells were treated with 50 nM LMAN1 siRNA or non-targeting (NT) siRNA using HiPerfect transfection reagent (Qiagen).

Fu Y.L., Zhang B, & Mu T.W. (2019). LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors. Biochemical and biophysical research communications, 511(2), 356-362.

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Comprehensive Cell Line Evaluation

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Irradiated 3T3msCD40L cells were used in single B cell culture assay. TZM-bl cells (NIH AIDS Reagents Program) were used in pseudovirus neutralization assay. Human HEK293T cells (ATCC) were used for pseudovirus production. FreeStyle HEK293 cells (ThermoFisher) was used for recombinant trimer production and monoclonal antibody production, CEM.NKR-CCR5-sLTR-Luc cells were used in ADCC assay.

Zhao F., Joyce C., Burns A., Nogal B., Cottrell C.A., Ramos A., Biddle T., Pauthner M., Nedellec R., Qureshi H., Mason R., Landais E., Briney B., Ward A.B., Burton D.R, & Sok D. (2020). Mapping Neutralizing Antibody Epitope Specificities to an HIV Env Trimer in Immunized and in Infected Rhesus Macaques. Cell Reports, 32(10), 108122.

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HEK 293T Cell Culture and Transfection

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Human HEK 293T cells (ATCC) were maintained in DMEM (Gibco) supplemented with 10% Heat-inactivated FBS (Gibco) and 1% penicillin/streptomycin (Gibco) in a 37 °C and 5% CO₂ incubator. Cells were confirmed to be free of mycoplasma by analyzing supernatant media monthly using MycoAlert^™ PLUS (Lonza). For HT-PAMDA experiments, transfections were performed between 16 and 20 hours after seeding 150,000 cells per well in a 24-well tissue culture treated plate (Corning). Approximately 600-700 ng of Cas expression vector (Supplementary Table 8) were combined with 1.5 μL of TransIT-X2 (Mirus) into a total volume of 50 μL including Opti-MEM (Thermo Fisher). Reagent mixture was gently vortexed, incubated at room temperature for 15 minutes, and added to cells with gentle shaking of the plate.

Conway J.F., Cheng N., Zlotnick A., Stahl S.J., Wingfield P.T, & Steven A.C. (1998). Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy. Proceedings of the National Academy of Sciences of the United States of America, 95(25), 14622-14627.

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Antiviral Effects of Type I and III IFNs

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Human HEK293T cells (ATCC catalog no. CRL-11268) were purchased and preserved in our lab. Cell culture was performed as previously described (Zhou et al., 2022 (link)). EV71 CC063 was isolated and preserved by our lab (Xu et al., 2020 (link)). Viruses were ampliﬁed using RD cells. Viral titers in cultural supernatants were determined by plaque assay.
Recombinant human IFN-β1b, IFN-γ, and IL-29 were purchased from BBI LIFE SCIENCES CORPORATION, Shanghai, China (C600038, C600039, C600117). The used concentration of IFNs was 250 U/mL for IFN-β1b, 250 U/mL for IFN-γ, and 250 U/mL for IL-29).

Zheng B., Zhou X., Tian L., Wang J, & Zhang W. (2022). IFN-β1b induces OAS3 to inhibit EV71 via IFN-β1b/JAK/STAT1 pathway. Virologica Sinica, 37(5), 676-684.

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Culturing Diverse Microbial and Mammalian Cells

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M. luteus (ATCC 4698) and S. aureus (ATCC 25923) were cultured in tryptic soy broth. GFP-E. coli was generated as described^{32 (link)} and cultured in LB medium. Drosophila S2 cells (RRID: CVCL_TZ72) and S2R+ cells (RRID: CVCL_Z831) were cultured at 25 °C in Drosophila Schneider’s Medium (Biological Industries) supplemented with 10% FBS and antibiotics. Human HEK293T cells (ATCC#CRL-11268) and mouse MEF cells (ATCC#CRL-2991) were cultured at 37 °C in DMEM medium (Sigma) supplemented with 10% FBS and antibiotics. Human THP-1 cells (ATCC#TIB-202) were cultured at 37 °C in RPMI 1640 medium (Sigma) supplemented with 10% FBS and antibiotics.

Yang Y., Zhou H., Huang X., Wu C., Zheng K., Deng J., Zheng Y., Wang J., Chi X., Ma X., Pan H., Shen R., Pan D, & Liu B. (2024). Innate immune and proinflammatory signals activate the Hippo pathway via a Tak1-STRIPAK-Tao axis. Nature Communications, 15, 145.

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