Hek293t cells

Manufactured by Leibniz Institute DSMZ

Sourced in Germany

HEK293T cells are a commonly used human embryonic kidney cell line derived from human embryonic kidney cells. They are widely used in cell biology research and biotechnology applications due to their high transfection efficiency and ability to produce recombinant proteins.

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HEK293T (26 citations)

23 protocols using hek293t cells

Optimized Lentiviral Particles for Neuronal Transduction

Cited 1 time

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Codon-improved Cre recombinase (iCre) (Shimshek et al., 2002 (link)) expressing lentiviral particles were generated as previously described (Kuhn et al., 2010 (link); Colombo et al., 2013 (link)). Briefly, lentiviruses were generated by transient cotransfection of HEK293T cells (DSMZ, Braunschweig, Germany) with the plasmids psPAX2, pCDNA3.1(−)-VSV-G and as transfer vector F2UΔZeo-iCre using Lipofectamine 2000 (Thermo). Lentiviral particles for infection of murine primary cortical neurons were concentrated and purified by ultracentrifugation. Lentiviral stocks were stored at −80°C until use.

Kuhn P.H., Colombo A.V., Schusser B., Dreymueller D., Wetzel S., Schepers U., Herber J., Ludwig A., Kremmer E., Montag D., Müller U., Schweizer M., Saftig P., Bräse S, & Lichtenthaler S.F. (2016). Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. eLife, 5, e12748.

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Generating Lentiviral Particles with AF-CAR

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HEK293T cells (#ACC 635; DSMZ, Brunswick, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. AF-CAR containing LV particles were generated by transfection of 1 × 10⁷ HEK293T cells with AF-CAR pDNA using the Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, USA). The viral supernatant of LV-CAR-transfected HEK293T cells was collected daily for 3 days. The pool of viral particles was concentrated using the Lenti-X concentrator (Takara Bio USA Inc., Mountain View, USA). The transduction efficiency and AF-CAR viral titers were estimated by serial dilution transduction of AF-CAR lentiviral particles with HEK293T cells as described before (15 (link)). AF-CAR LV particles were aliquoted and cryopreserved at −80°C.

Kumaresan P.R., Wurster S., Bavisi K., da Silva T.A., Hauser P., Kinnitt J., Albert N.D., Bharadwaj U., Neelapu S, & Kontoyiannis D.P. (2024). A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus. mBio, 15(4), e03413-23.

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Genetic Modification of Airway Organoids

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Airway organoids were cultured in growth medium at 37°C, 5% CO_2, and a humidified atmosphere as described above. Medium was changed every 2–3 days and organoids were passaged every 10–14 days. Genetic modification of organoid cells, allowing the expression of a fluorescent protein, was achieved via lentiviral transduction. Lentiviral particles were produced via third-generation split packaging protocol in HEK293T cells (DSMZ, Germany) as previously described (Schambach et al., 2006 (link)). The transfer plasmid contained the sequence for expression of a histone 2A-mCherry fusion protein under control of a Trp63 promoter, which was amplified from human genomic DNA as previously described (Lanza et al., 2006 (link)). Lentiviral transduction was performed based on a protocol previously described for employment in intestinal organoids by van Lidth de Jeude and colleagues (Van Lidth De Jeude et al., 2015 (link)).

Gentemann L., Donath S., Seidler A.E., Patyk L., Buettner M., Heisterkamp A, & Kalies S. (2023). Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids. Frontiers in Cell and Developmental Biology, 11, 1268621.

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Lentiviral production and AML cell culture

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HEK293T cells (DSMZ, Germany, catalog no.: Acc635), used for lentivirus production, were obtained from DSMZ and cultivated in Dulbecco’s modified Eagle’s medium (Biowest, catalog no.: L0104) supplemented with 10% fetal calf serum (Catus Biotech, catalog no.: BS- 2020-500). All OCI-AML (Luc⁺/GFP⁺) cell lines (DSMZ, Germany, catalog no.: ACC 582, ACC 99) were cultured in minimum essential medium α (PAN-Biotech, catalog no.: P04-21250) supplemented with 20% fetal calf serum.

Teppert K., Yonezawa Ogusuku I.E., Brandes C., Herbel V., Winter N., Werchau N., Khorkova S., Wöhle C., Jelveh N., Bisdorf K., Engels B., Schaser T., Anders K., Künkele A, & Lock D. (2024). CAR’TCR-T cells co-expressing CD33-CAR and dNPM1-TCR as superior dual-targeting approach for AML treatment. Molecular Therapy Oncology, 32(2), 200797.

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Patch-Clamp Experiments on Transfected HEK 293T Cells

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HEK 293T cells (DSMZ, Braunschweig, Germany) were maintained in DMEM/F-12 (Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO₂ incubator. Cells were trypsinized, diluted with culture medium, and seeded on 12-mm glass coverslips. Patch-clamp experiments were performed 2–3 days after plating. Cells were transfected with the respective plasmids using the Rotifect^® (Roth, Karlsruhe, Germany) transfection reagent. CD8-encoding plasmids (10–20% of total DNA) were co-transfected to allow identification of transfected cells using anti-CD8-coated beads (Dynabeads, Invitrogen, Karlsruhe, Germany).

Gessner G., Sahoo N., Swain S.M., Hirth G., Schönherr R., Mede R., Westerhausen M., Brewitz H.H., Heimer P., Imhof D., Hoshi T, & Heinemann S.H. (2017). CO-independent modification of K+ channels by tricarbonyldichlororuthenium(II) dimer (CORM-2). European journal of pharmacology, 815, 33-41.

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Culturing HEK293T and NALM-6 Cells

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HEK293T cells were obtained from DSMZ. They were cultured in DMEM (Life Technologies) with 10% fetal bovine serum (Life Technologies) and 1% l-glutamine (Life Technologies). NALM-6 cells were obtained from ATCC and cultured in RPMI medium with the same additives as for HEK293T cells. All cells were kept at 37 °C in a humidified incubator containing 5% CO₂. They were routinely tested for mycoplasma infection. Viably-cryopreserved cells from a patient-derived xenograft model of human B-ALL harboring a TCF3-HLF fusion (ALL1807) were established as previously described [17 (link)] and used for downstream sequencing studies.

Schulz L., Torres-Diz M., Cortés-López M., Hayer K.E., Asnani M., Tasian S.K., Barash Y., Sotillo E., Zarnack K., König J, & Thomas-Tikhonenko A. (2021). Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts. Genome Biology, 22, 190.

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Breast Cancer Cell Line Maintenance

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The breast cancer cell lines BT549, HCC1806, MDA-MB-231, MDA-MB-468, MCF7, MDA-MB-361, SKBR3, and T47D (acquired from ATCC, Gaithersburg, MA, USA or DSMZ GmbH, Braunschweig, Germany collections) were maintained in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FCS and non-essential amino acids (Sigma Aldrich, St. Lois, MO, USA) when necessary or in RPMI 1640 (Roswell Park Memorial Institute) medium supplemented with 10% FCS and human insulin (10 µg/mL). The human embryonic kidney HEK293T cells (from DSMZ GmbH collection) were used for lentivirus productions and grown in DMEM medium with 10% FCS. Cultivation was performed under standard conditions in water humified 37 °C incubator with 5% CO₂, either for 2D or 3D cell analysis. Cell lines were checked for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd., Basel, Switzerland) and their identity verified by genetic profiling using the PowerPlex^® 16 System (Eurofins/MWG Operon, Munich, Germany).

Anastasov N., Hirmer E., Klenner M., Ott J., Falkenberg N., Bao X., Mutschelknaus L., Moertl S., Combs S., Atkinson M.J, & Schmid T. (2020). MEK1 Inhibitor Combined with Irradiation Reduces Migration of Breast Cancer Cells Including miR-221 and ZEB1 EMT Marker Expression. Cancers, 12(12), 3760.

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Cell Culture Conditions for KBM-7 and HEK293T

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KBM-7 cells were cultured in IMDM supplemented with 10% fetal calf serum FCS (PAN-Biotech), 100 U/ml penicillin, 100 μg/ml streptomycin (PAN-Biotech) and L-glutamine (PAN-Biotech). HEK293T cells (DSMZ, Germany) were grown in DMEM high glucose supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (PAN-Biotech).

Förster B., Demangel C, & Thye T. (2020). Mycolactone induces cell death by SETD1B-dependent degradation of glutathione. PLoS Neglected Tropical Diseases, 14(10), e0008709.

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HEK293T and ST2 Cell Culture

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HEK293T cells (DSMZ, Braunschweig, Germany) were cultured in DMEM GlutaMAX medium (Gibco, Waltham, MA, USA), ST2 cells (DSMZ)^{25 (link)} in RPMI 1640 GlutaMAX medium (Gibco) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin/Streptomycin (Gibco). The cells were cultured at 37 °C in a 5% CO₂ atmosphere. At regular intervals, the cell culture was tested as mycoplasma-free. For Prmt6 inhibition, the selective inhibitor SGC6870 (Sigma, Darmstadt, Germany) and the control compound SGC6870N were used in a concentration of 5 µM^{26 (link)}. For Prmt6 overexpression and knockout experiments, ST2 cells were transduced with Prmt6-LeGOiG2 or gRNA’s in lentiCRISPRv2 vector. Generation and production of virus was done as described^{27 (link)}. LeGOiG2 empty vector and a vector expressing non-specific gRNA served as controls, respectively. The gRNA sequences are listed in the Supplemental Material Table 1.

Gerstner M., Heller V., Fechner J., Hermann B., Wang L, & Lausen J. (2024). Prmt6 represses the pro-adipogenic Ppar-gamma–C/ebp-alpha transcription factor loop. Scientific Reports, 14, 6656.

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Malignant Meningioma Cell Line Protocol

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The cell lines used were generated from the malignant meningioma cell line IOMM-Lee and cultured as previously described [25 (link)]. HEK293T cells were purchased from DSMZ (Braunschweig, Germany).

von Spreckelsen N., Waldt N., Poetschke R., Kesseler C., Dohmen H., Jiao H.K., Nemeth A., Schob S., Scherlach C., Sandalcioglu I.E., Deckert M., Angenstein F., Krischek B., Stavrinou P., Timmer M., Remke M., Kirches E., Goldbrunner R., Chiocca E.A., Huettelmaier S., Acker T, & Mawrin C. (2020). KLF4K409Q–mutated meningiomas show enhanced hypoxia signaling and respond to mTORC1 inhibitor treatment. Acta Neuropathologica Communications, 8, 41.

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