Hek293t cells

Manufactured by RIKEN BioResource Center

Sourced in Japan

HEK293T cells are a human embryonic kidney cell line that are commonly used in cell biology research and biopharmaceutical applications. These cells are highly transfectable and can be used for the production and expression of recombinant proteins.

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HEK293T cells (RBC2202) HEK293T

23 protocols using hek293t cells

Human Cell Line Cultivation and Neutrophil Isolation

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HEK293T cells, a non-cancerous human embryonic kidney epithelial cell line with a stable expression of the SV40 large T antigen, were obtained from the RIKEN BioResource Center (Tsukuba, Japan). Human pancreatic cancer PANC-1, AsPC-1, and MIA PaCa-2 cells, human breast cancer MDA-MB-231 and MCF-7 cells, human prostate cancer PC-3 and LNCaP cells, human squamous cancer A431 cells, human cervical cancer HeLa cells, human melanoma MeWo cells, human mesothelioma MSTO-211H cells, and human lung cancer NCI-H2170 cells were obtained from the American Type Culture Collection (Rockville, MD). All cell lines were cultivated in DMEM/F12 (D/F) medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS).
Human neutrophils were isolated from the blood of a healthy donor by the conventional method. In brief, the polymorphonuclear cell fraction was first collected from the whole blood by centrifugation at 2000 rpm for 30 min using the PolymorphPrep™ solution (Serumwerk Bernburg, Bernburg, Germany). The collected cells were then incubated with CD16 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and the unbound flow through the fraction was collected as the neutrophil-enriched fraction.

Gohara Y., Tomonobu N., Kinoshita R., Futami J., Audebert L., Chen Y., Komalasari N.L., Jiang F., Yoshizawa C., Murata H., Yamamoto K.I., Watanabe M., Kumon H, & Sakaguchi M. (2023). Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells. Journal of Molecular Medicine (Berlin, Germany), 101(4), 431-447.

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Cell Culture and Lipid Nanoparticle Preparation

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Human embryonic kidney (HEK) 293T cells, mouse melanoma B16-F10 cells, and mouse embryonic fibroblast BALB/3T3 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu City, Taiwan, ROC). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Gaithersburg, MD, USA) supplemented with inactivated 10% foetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin amphotericin B (PSA; Biological industries, New York, NY, USA). Human natural killer NK-92 MI cells were grown in alpha minimum essential medium (αMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.2 mM inositol (Sigma-Aldrich), 0.2 mM 2-mercaptoethanol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), 12.5% horse serum (Gibco BRL, Gaithersburg, MD, USA) and 12.5% FBS. All cells were incubated at 37 °C in an atmosphere of 5% CO₂.
Male C57BL/6 mice (6–8 weeks old) were purchased from the National Laboratory Animal Center (NLAC, Taipei City, Taiwan, ROC).
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Avanti Polar Lipids, Inc.) and polyethylene glycol (PEG, MW 1.500) (Shimo-Meguro, Meguro-Ku, Tokyo, Japan), PEG (MW 8.000, Sigma-Aldrich) and polyethylenimine (PEI, branched, MW 25.000, Sigma-Aldrich) were purchased to prepare LPPC.

Ho S.Y., Chen P.R., Chen C.H., Tsai N.M., Lin Y.H., Lin C.S., Chuang C.H., Huang X.F., Chan Y.L., Liu Y.K., Chung C.H., Weng S.L, & Liao K.W. (2020). Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules. Journal of Nanobiotechnology, 18, 58.

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Cloning and Expression Vectors for ARL3 and PDE6D

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cDNAs for human ARL3 (NM_004311) and PDE6D (NM_002601) were obtained from a cDNA library by PCR amplification. Expression vectors used in this study are listed in Table S1; some of them were constructed in our previous studies (Nozaki et al., 2017 (link); Qiu et al., 2021 (link)). The antibodies used in this study are listed in Table S2. GST-tagged anti-GFP Nb prebound to glutathione-Sepharose 4B beads were prepared as described previously (Katoh et al., 2015 (link); 2018 (link)). Polyethylenimine Max and SAG were purchased from Polysciences and Enzo Life Sciences, respectively. HEK293T cells and hTERT-RPE1 cells were obtained from RIKEN BioResource Research Center (RBC2202) and American Type Culture Collection (CRL-4000), respectively.

Fujisawa S., Qiu H., Nozaki S., Chiba S., Katoh Y, & Nakayama K. (2021). ARL3 and ARL13B GTPases participate in distinct steps of INPP5E targeting to the ciliary membrane. Biology Open, 10(9), bio058843.

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Cell Culture and Transfection Protocols

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HEK293T cells (RIKEN BRC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS, Biowest) at 37 °C in humidified air containing 5% CO₂. K562 cells (RIKEN BRC) were maintained in Iscove’s modified Dulbecco’s medium (IMDM, FUJIFILM) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific) and 50 μg/mL kanamycin at 37 °C in humidified air containing 5% CO₂. HEK293T cells were transfected using polyethylenimine “MAX” (Cosmo Bio). K562 cells were electroporated using an Amaxa 4D-Nucleofector (Lonza).

Kanadome T., Hoshino N., Nagai T., Matsuda T, & Yagi T. (2021). Development of FRET-based indicators for visualizing homophilic trans interaction of a clustered protocadherin. Scientific Reports, 11, 22237.

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Lentiviral Transduction of Trophoblast Stem Cells

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Lentiviral vectors were cotransfected with the packaging plasmids into HEK293T cells purchased from RIKEN BRC (RCB2202). After incubation, the supernatants were collected twice at days 2 and 3, passed through a 0.45-μm filter, and centrifuged at 8000g for at least 14 h at 4°C. The virus pellets were suspended in TSC medium and mixed with the single-cell suspensions of TSCs derived by crossing C57BL/6NCrSlc female mice with JF1 male mice. After incubation for 4 h, the cells were washed and seeded onto fibronectin-coated dishes.

Hada M., Miura H., Tanigawa A., Matoba S., Inoue K., Ogonuki N., Hirose M., Watanabe N., Nakato R., Fujiki K., Hasegawa A., Sakashita A., Okae H., Miura K., Shikata D., Arima T., Shirahige K., Hiratani I, & Ogura A. (2022). Highly rigid H3.1/H3.2–H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells. Genes & Development, 36(1-2), 84-102.

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Maintenance of Mouse Spermatogonial Stem Cells

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For SSC cultures, Thy1⁺ germ cells were collected from DBA/2 and BDF mouse pups and maintained as described previously^{66 (link)}. Briefly, the germ cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in SSC culture medium, which consisted of basal culture medium for SSCs supplemented with Knockout Serum Replacement (10% for DBA/2 SSCs; 2% for BDF SSCs and testicular cells)(Gibco, Carlsbad, CA), 0.2% (w/v) BSA (MP Biochemicals, Santa Ana, CA), and 20 ng/ml human GDNF, unconjugated (Peprotech, Rocky Hill, NJ). While BDF strain cells were subjected to the transplantation experiments, DBA/2 were to the other experiments. For the experiments of GDNF depletion, the cultured DBA/2 strain SSCs were washed three times and cultured in GDNF-free SSC culture medium for the indicated periods. HEK293T cells were purchased from RIKEN Bioresource Research Center (RCB2202).

Makino Y., Jensen N.H., Yokota N., Rossner M.J., Akiyama H., Shirahige K, & Okada Y. (2019). Single cell RNA-sequencing identified Dec2 as a suppressive factor for spermatogonial differentiation by inhibiting Sohlh1 expression. Scientific Reports, 9, 6063.

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Harringtonin Pulse-Chase in HEK293T

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HEK293T cells (RIKEN BRC, Ibaraki, Japan) (approximately 5.0 × 10 5 cells) were seeded in 6-well dishes coated with collagen (Cellmatrix Type I-C; Nitta Gelatin, Osaka, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai) supplemented with 10% fetal bovine serum (NICHIREI, Tokyo, Japan; Cat. No. 175012, Lot. No. 20C00F) at 37 °C under 5% CO2. After 24 h, transfection was performed using polyethylenimine (PEI) Max (Polysciences, Warrington, USA) with 1.25 µg of plasmid DNA. After a further 24 h, the cells were collected for analysis. For the harringtonin pulse-chase experiments, 1 µg/mL harringtonin was added to the HEK293T cells after 22 h of translation, and their lysates were recovered at the indicated times.

Sogawa A., Komori R., Yanagitani K., Ohfurudono M., Tsuru A., Kadoi K., Kimata Y., Yoshida H, & Kohno K. (2023). Signal sequence-triage is activated by translocon obstruction sensed by an ER stress sensor IRE1α. Cell structure and function, 48(2).

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Transfection of P2X2 in HEK293 Cells

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HEK293T cells (RIKEN Bio‐Resource Center, Tsukuba, Japan) were cultured in HEK medium consisting of Dulbecco's modified Eagle's medium (DMEM; Sigma‐Aldrich, Taufkirchen, Germany) containing 10% FBS (Biowest, Nuaillé, France), penicillin (100 U·mL⁻¹), and streptomycin (100 µg·mL⁻¹; P/S; Nacalai Tesque Inc., Kyoto, Japan). HEK293 cells stably expressing murine choline acetyltransferase (ChAT; ChAT‐HEK293 cells) [29 (link)] were cultured in ChAT‐HEK medium consisting of DMEM containing 10% FBS and hygromycin B (500 µg·mL⁻¹; Nacalai Tesque, Inc., Kyoto, Japan). HEK293T or ChAT‐HEK293 cells were plated on 10‐cm dishes (2 × 10⁶ cells per dish) maintained at 37 °C in a 5% CO₂/air atmosphere. HEK293T or ChAT‐HEK293 cells were transfected with the pP2X₂‐IRES2‐AcGFP1‐Nuc vector (10 μg plasmid DNA per dish) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and transfected cells were incubated for 2 days.

Maruyama T., Mano A., Ishii T., Kakinuma Y, & Kaneda M. (2021). P2X2 receptors supply extracellular choline as a substrate for acetylcholine synthesis. FEBS Open Bio, 12(1), 250-257.

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Expression Vectors for Protein Studies

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cDNAs for human CFAP20 (NM_013242.3) and FAM149B1 (NM_173348.2) were obtained from a cDNA library by PCR amplification. Human ICK cDNA was kindly provided by Takahisa Furukawa (Osaka University) (Chaya et al., 2014 (link)). Expression vectors for BROMI, CFAP20, FAM149B1, CCRK, and ICK and their deletion and point mutants and vectors for the production of lentiviruses expressing them are listed in Supplemental Table S1. Several of the vectors were constructed in our previous studies (Hamada et al., 2018 (link); Nakamura et al., 2020 (link); Noguchi et al., 2021 (link)). Antibodies used in this study are listed in Supplemental Table S2. GST-tagged anti-GFP Nb and anti-mChe Nb (the LaM-2 version) prebound to glutathione–Sepharose 4B beads were prepared as described previously (Katoh et al., 2015 (link); Katoh et al., 2018 (link); Ishida et al., 2021 (link)). SAG and Polyethylenimine Max were purchased from Enzo Life Sciences and Polysciences, respectively. HEK293T cells and hTERT-RPE1 cells were obtained from RIKEN BioResource Research Center (Catalogue No. RBC2202) and American Type Culture Collection (Catalogue No. CRL-4000), respectively.

Satoda Y., Noguchi T., Fujii T., Taniguchi A., Katoh Y, & Nakayama K. (2022). BROMI/TBC1D32 together with CCRK/CDK20 and FAM149B1/JBTS36 contributes to intraflagellar transport turnaround involving ICK/CILK1. Molecular Biology of the Cell, 33(9), ar79.

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Culturing Human Cell Lines

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The human MRT cell lines MP-MRT-AN, KP-MRT-RY, and KP-MRT-YM were established as previously reported [16] [17] [18] . They were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS). HEK293T cells were purchased from RIKEN BRC, Japan and maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and 1% PS. Cells were cultured at 37 °C and 5% CO 2 .

Daifu T., Mikami M., Hiramatsu H., Iwai A., Umeda K., Noura M., Kubota H., Masuda T., Furuichi K., Takasaki S., Noguchi Y., Morita K., Bando T., Hirata M., Kataoka T.R., Nakahata T., Kuwahara Y., Iehara T., Hosoi H., Takita J., Sugiyama H., Adachi S, & Kamikubo Y. (2021). Suppression of malignant rhabdoid tumors through Chb-M'-mediated RUNX1 inhibition. Pediatric blood & cancer, 68(2).

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