Hek293t

Manufactured by Polysciences

Sourced in United States

HEK293T is a cell line derived from human embryonic kidney cells. It is commonly used in biological research and biotechnology applications due to its ability to efficiently produce recombinant proteins and viral particles.

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16 protocols using hek293t

Cell Culture and Transfection Protocols

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Human embryonic kidney cells (HEK293T), human myeloid leukemia mononuclear cells (THP-1), human hepatoma cells (Huh-7), mouse hepatocarcinoma cells (Hepa1-6), mouse mononuclear macrophages cells (Raw264.7) and mouse fibroblast cells (L929) were purchased from the China Center Type Culture Collection. HEK293T, Huh-7, Hepa1-6, Raw264.7, and L929 cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL). THP-1 cells were cultured in RPMI1640 (Thermo Fisher Scientific, Waltham, USA) containing 10% FBS and 1% penicillin-streptomycin. All cells were grown in a 37°C cell culture incubator containing 5% CO₂.
For transfection of HEK293T, the cells were inoculated at 80% in the indicated culture dishes and used polyethylenimine (Polysciences, Philadelphia, USA) according to the manufacturer’s protocol. For macrophage cell lines, Raw264.7 and THP-1 cells were transfected with liposomal transfection reagent (Yeasen, Shanghai, China).

Yu C., Zhu Q., Ma C., Luo C., Nie L., Cai H., Wang Q., Wang F., Ren H., Yan H., Xu K., Zhou L., Zhang C., Lu G., Lu Z., Zhu Y, & Liu S. (2024). Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6. Frontiers in Immunology, 14, 1289795.

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Transfection of Cell Lines

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HEK 293T, HeLa, BOSC23 and MDA-MB-468 cell lines were used in this work. All cell lines were purchased from American Type Culture Collection, which were tested and authenticated by the cell bank using their standard short tandem repeats (STR)-based techniques. Cells were also continuously tested for mycoplasma contamination by using 4',6-diamidino-2-phenylindole (DAPI) staining. HEK 293T or HeLa were transfected with various plasmids using PEI (Polysciences) according to the manufacturer's protocol. Briefly, the plasmid diluted in serum-free RPMI medium was mixed with PEI (1 μg μl⁻¹) in 1:3 ratio. After incubating the DNA–PEI mixture at room temperature (RT) for 15 min, the complexes were added to cells to allow the transfection of plasmid.

Shinde S.R, & Maddika S. (2016). PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7. Nature Communications, 7, 10689.

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Cell Line Transfection and Murine Spleen Isolation

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Namalwa (human Burkitt B-cell lymphoma), Cos7 (African green monkey kidney), HEK293T (human embryonal kidney) cell lines were obtained from the American Type Culture Collection (ATCC) and cultivated as previously described [9 (link)].
Transient transfections of Cos7 cells were performed using polyethylenimine (PEI) 25K (Polyscience, Inc) according to the manufacturer’s instructions and HEK293T cells were transfected with the standard calcium-phosphate precipitation method. The hematopoietic cells were transfected by electroporation using a Neon Electroporator (Life Technologies) and the 100μL tip kit with the following settings: 1350V (pulse-voltage); 20ms (pulse-width); 2 (pulse-number). Single cell suspensions were prepared from spleens obtained from normal and Btk knock-out (KO) mice. The use of mouse models in this study was approved by Stockholm South Animal Ethics Committee with registration number S56-14.

Gustafsson M.O., Mohammad D.K., Ylösmäki E., Choi H., Shrestha S., Wang Q., Nore B.F., Saksela K, & Smith C.I. (2017). ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library. PLoS ONE, 12(4), e0174909.

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HEK293T Cell Culture and Transfection

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HEK293T cells were purchased from Life technologies and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cell lines, were maintained at 37°C and in a 5% CO₂ controlled atmosphere. HEK293T were transfected with plasmid DNA using polyethylenimine‎ (Polysciences, Warrington, PA, USA) according to the manufacturer's recommendations.

Civiero L., Cirnaru M.D., Beilina A., Rodella U., Russo I., Belluzzi E., Lobbestael E., Reyniers L., Hondhamuni G., Lewis P.A., Van den Haute C., Baekelandt V., Bandopadhyay R., Bubacco L., Piccoli G., Cookson M.R., Taymans J, & Greggio E. (2015). Leucine‐rich repeat kinase 2 interacts with p21‐activated kinase 6 to control neurite complexity in mammalian brain. Journal of Neurochemistry, 135(6), 1242-1256.

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Viral Production in HEK293T Cells

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Human embryonic kidney 293T cells (HEK293T) was procured from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM medium (Hyclone, Logan, UT, USA, #SH30243) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA, #SH30070) and 1% antibiotic/antimycotic (Hyclone, Logan, UT, USA, #SV30010). DNA transfections were done with BCLxl constructs using polyethylenimine (Polysciences, Warrington, PA, USA, #23966-2) in HEK293T cells and viruses were collected as described [23 (link)].

Saurabh K., Scherzer M.T., Song A., Yip K.W., Reed J.C., Li C, & Beverly L.J. (2014). Dissecting the in vivo leukemogenic potency of BCLxl. Journal of leukemia (Los Angeles, Calif.), 2(5), 158-.

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Transfection and Retroviral Infection Protocols

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HEK293T and 786-O cell lines were purchased from the American Type Culture Collection and cultured as previously described [11 ]. HEK293T cells were transfected with the expression plasmid, using polyethyleneimine (PEI) (MW-25K, Polyscience Inc., Warrington, PA, USA), plasmid DNA (μg):PEI (μg) = 1:3. Retroviral infections were performed as described previously [28 (link)]. In brief, 786-O cells were incubated in retrovirus-containing culture medium for 2 days and selected using puromycin (5 μg/ml) for 1 week. Additionally, siRNA was transfected into 786-O cells, using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) per the manufacturer’s instructions. At 2 days post transfection, cells were harvested and lysed.

Okumura F., Joo-Okumura A., Nakatsukasa K, & Kamura T. (2017). Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2. PLoS ONE, 12(4), e0175593.

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Cell Culture and Treatment Protocols

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The following cell lines, namely HEK293T, PC-3, and DU145, were procured from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China): HEK293T and DU145 cells were cultivated in Dulbecco’s modified eagle medium (DMEM; SH30022.01, Gibco, USA), whereas PC-3 cells were maintained in RPMI-1640 medium (C11875500BT, Gibco, USA). The incubation conditions were maintained at 37 °C with a 5% CO₂ atmosphere. The culture media were supplemented with 10% fetal bovine serum (FBS; A3160802, Gibco, USA) and 1% penicillin–streptomycin solution (15140-122, Gibco, USA). For transfection experiments, the HEK293T, PC-3, and DU145 cells were transfected with the designated plasmids utilizing the polyethyleneimine (PEI; 23966-1, Polysciences, USA) technique. With regard to cellular treatment, PC-3 cells underwent serum starvation in RPMI-1640 medium overnight, followed by treatment with JNK-IN-8 (1 μmol/L and 2 μmol/L, HY-13319, MCE, USA) and bafetinib (5 μmol/L, HY-50868, MCE, USA) for specified durations. All cultured cells were subjected to routine mycoplasma contamination screening via DNA detection method.

Weng H., Xiong K.P., Wang W., Qian K.Y., Yuan S., Wang G., Yu F., Luo J., Lu M.X., Yang Z.H., Liu T., Huang X., Zheng H, & Wang X.H. (2023). Aspartoacylase suppresses prostate cancer progression by blocking LYN activation. Military Medical Research, 10, 25.

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Generation of Gene-Edited HEK293T Cells

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HEK293T cells (American Type Culture Collection CRL-
3216) were grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum, 2 mM l-glutamine, penicillin (5 U/ml), and streptomycin (50 μg/ml). To introduce expression vectors, HEK293T cells were transfected with
PEI MAX (Polysciences Inc., Warrington, PA, USA). UFL1
(5′-CCAGCGGGCGCAGTTCGCCG-3′) or UFBP1 (5′-GTAGCGGCGGCTCTGCTAGT′) guide RNA was designed using the CRISPR Design tool (https://crispr.dbcls.jp/) and subcloned
into pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene, #42230), a human codon–optimized SpCas9 and chimeric guide RNA expression plasmid. To generate UFL1, UFC1, CDK5RAP3, and UFBP1 UFSP2 KO HEK293T cells, HEK293T or UFSP2 KO HEK293T cells (16 (link)) were transfected the aforementioned pX330 vectors together with pEGFP-C1 (#6084-1, Clontech Laboratories, Mountain View, CA, USA) and cultured for 2 days. GFP-positive cells were sorted and expanded. Ablation of UFL1, UFC1, or CDK5RAP3 was confirmed by a heteroduplex mobility assay followed by immunoblot analysis with anti-UFL1, anti-UFC1, or anti-CDK5RAP3 antibody. UFM1- (23 (link)), UFC1- (23 (link)), UFBP1- (27 (link)), CDK5RAP3- (27 (link)), and UFSP2-deficient HEK293T cells (16 (link)) were used in this study. HEK293T and HeLa cells were authenticated by short-tandem repeat profile. All cell lines were tested for mycoplasma contamination.

Ishimura R., Ito S., Mao G., Komatsu-Hirota S., Inada T., Noda N.N, & Komatsu M. (2023). Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control. Science Advances, 9(33), eadh3635.

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Cell Line Culture and Transfection

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COS-7 (African green monkey fibroblast-like kidney), HEK-293T (human embryonic kidney cells) and DT40 (chicken lymphoma cells) were obtained from the American Type Culture Collection. The B7.10 cell line (DT40 chicken lymphoma cells in which the BTK gene is inactivated) was generated in Dr T. Kurosaki's laboratory,^{5 (link)} and has been characterized in great detail previously.^{39 (link)} COS-7 and HEK-293T cells were cultured in Dulbecco's modified eagle medium supplemented with 10% heat-inactivated fetal bovine serum. DT40 and B7.10 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 5% chicken serum, 5% glutamine, 50 μm 2-mercaptoethanol and penicillin/streptomycin. Cells were grown at 37 °C under 5% CO₂. COS-7 and HEK-293T cells were transiently transfected in six-well plates using polyethylenimine (Polyscience, Inc., Warrington, PA, USA). B7.10 cells were transfected using the Neon transfection system (Life technologies, La Jolla, CA, USA).

Hamasy A., Wang Q., Blomberg K.E., Mohammad D.K., Yu L., Vihinen M., Berglöf A, & Smith C.I. (2016). Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant. Leukemia, 31(1), 177-185.

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Cell Culture and Transfection Protocol

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HeLa, HCT116, and RKO cells were purchased from ATCC and HEK293T was acquired from National Infrastructure of Cell Line Resource. HeLa, HCT116 and HEK293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C with 5% CO₂. RKO cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C with 5% CO₂. HeLa, HCT116, RKO and HEK293T cells were transfected with PEI according to the manufacturer's instructions (Polyscience).

Fu J., Zhou S., Xu H., Liao L., Shen H., Du P, & Zheng X. (2023). ATM–ESCO2–SMC3 axis promotes 53BP1 recruitment in response to DNA damage and safeguards genome integrity by stabilizing cohesin complex. Nucleic Acids Research, 51(14), 7376-7391.

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