Hek293t cells
Manufactured by Takara Bio
Sourced in United States, Japan
HEK293T cells are a widely used cell line derived from human embryonic kidney cells. They are transformed with the adenovirus 5 DNA, which contains the SV40 large T antigen. HEK293T cells are known for their high transfection efficiency and are commonly used for the production and characterization of recombinant proteins, viral vector production, and various cell-based assays.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Puromycin, by Merck Group (3 mentions)
Hek293t cells, by Thermo Fisher Scientific (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Polybrene, by Merck Group (3 mentions)
Lenti x concentrator, by Takara Bio (3 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (2 mentions)
Xfect transfection reagent, by Takara Bio (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
D glucose, by Thermo Fisher Scientific (2 mentions)
Tet approved fbs, by Takara Bio (2 mentions)
Hek293t cells, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Digitonin, by Merck Group (2 mentions)
Anti flag, by Merck Group (2 mentions)
63 protocols using hek293t cells
1
Overexpression and Knockdown of SLC4A4/SLC4A5 in hRPTCs
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Overexpressed (OE): A Lentiviral construct (CCSB-Broad Lentiviral Expression Human SLC4A5 Clone; CloneId: ccsbBroad304_12409) was purchased from Thermo Scientific. The plasmid was packaged into virus with compatible packaging plasmids using HEK-293T cells (Clontech Laboratories). The lentivirus was added to hRTPCs at 30–40% confluence for 18–20 h, then removed and replaced with regular growth medium. After 48 h, the medium was changed to selection medium containing Blasticidin S (InvivoGen; 5 μg/ml).
Knocked-down (KD): Validated Mission shRNAi Lentiviral constructs for knocking down the SLC4A4 gene (Clone ID:NM_003759.1-598s1c1; NM_003759.1-1955s1c1 and NM_003759.1-3183s1c1) or SLC4A5 gene (Clone ID:NM_021196.3-676s21c1; NM_021196.3-1161s21c1 and NM_021196.2-1989s1c1), as well as a negative shRNA control (Mission Non-Target shRNA control vector; SHC002), were purchased from Sigma-Aldrich and packaged into virus with compatible packaging plasmids using HEK-293T cells (Clontech Laboratories). hRPTCs were transduced, as mentioned above. After 48 h, the selection agent puromycin (Sigma-Aldrich, P7255; 2μg/ml) was added to the media. KD of protein expression was confirmed by western blotting using an anti-NBCe2 antibody (Sigma HPA036621, polyclonal rabbit; 1:500 dilution). The western blots were analyzed using Odyssey software.
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2
Cultivation of Diverse Cell Lines
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Human liver epithelial THLE-2 cells (ATCC Cat#: CRL-2706) were cultured under standard conditions (37 °C and 5% CO2) in Bronchial Epithelial Cell Growth medium (Lonza Cat#: CC3170) supplemented with 10% FBS (Gibco Cat#: 10270106), all items included in the BulletKit (Lonza Cat#: CC4175), except for Gentamycin/Amphotericin (GA) and Epinephrine, 1% PSA (Thermo Fisher Cat#: 15240062), 1% glutamine (Thermo Fisher Cat#: 25030024), 5 ng/mL EGF (Thermo Fisher Cat#: PHG0315) and 70 ng/mL Phosphorylethanolamine (Sigma Aldrich Cat#: P0503). Vero E6 cells (ATCC Cat#: CRL-1586) were kindly provided by Nicola G.A. Abrescia (CIC bioGUNE) and cultured in MEM (Gibco Cat#: 31095-029) supplemented with 10% FBS (Thermo Fisher Cat#: 10270106) and 1% Penicillin-Streptomycin (P/S) (Thermo Fisher Cat#: 15140122). HEK293T cells (Takara Bio Inc. Cat#: 632180) were cultured in DMEM (Gibco Cat#: 41966-029) supplemented with 10% FBS and 1% P/S. Second-generation human upcyte hepatocytes, culture medium, high-performance medium, and thawing medium were all obtained from Upcyte Technologies (Heidelberg, Germany) and cultured as previously described68 (link).
3
Culturing HEK293T and Lenti-X 293T cells
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HEK293T cells and Lenti-X 293T cells (Takara, Shiga, Japan) were treated at 37°C in a 5% CO2 atmosphere to maintain saturation in Dulbecco’s modified Eagle’s Medium (DMEM) (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 5% FBS (Biowest, Nuaillé, France) and 100 U/mL of Antibiotic-Antimycotic Mixed (Nacalai tesque, Kyoto, Japan).
4
Isolation and Characterization of Human Bone Marrow Mesenchymal Stem Cells
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Human bone marrow aspirates were acquired from 3 cm posterior to the anterior superior iliac spine of fifteen adult donors. This research was authorized by the Institutional Review Board of Yonsei University College of Medicine (IRB no. 4-2017-0232). Label-free cell isolation was conducted based on biophysical characterization [41 (link)]. hBMSCs were isolated based on adhesion to a plastic culture plate surface, and their validity was verified by flow cytometry, as described in our previous study [38 (link)]. Subpopulations of hBMSC clones were characterized, and their multipotency was proven as previously described [39 (link)]. Additional hBMSCs were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured as previously described [16 (link)]. hBMSCs were cultured in Dulbecco’s Modified Eagle Medium Low-Glucose (DMEM-LG, 31600-034, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, 16000-044, Gibco) and 1% antibiotic-antimycotic solution (15240-062, Gibco). HEK293T cells were purchased from Takara Bio (Mountain View, CA, USA). HEK293T cells were maintained in DMEM High-Glucose (DMEM-HG, 12800-017, Gibco) containing 10% FBS and 1% antibiotic-antimycotic solution (Gibco), according to Takara Bio guidelines. Cells were incubated at 37 °C in a 5% CO2 atmosphere.
5
Engineered HEK293T Cell Line for GFP Expression
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We used HEK293T cells (Takara Bio, Japan). The identity and negative mycoplasma status of these cells have been confirmed by the vendors. Upon receiving the cell line, we engineered the cells to carry a genomically integrated dscGFP gene driven by the TRE3G promoter (consisting of seven repeats of the Tet response element) (Kempton et al., 2020 (link)). This cell line was clonally sorted and expanded and showed no background GFP fluorescence. We expanded and kept multiple aliquots in the liquid nitrogen freezer. The cells were taken out of the liquid nitrogen freezer for experiments and discarded after maximum 3 months of use.
6
Culturing Lung, Kidney, and HEK 293T Cell Lines
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The NCI-H460 human lung epithelial cell line was obtained from DSMZ (German Collection of Microorganisms and Cell Lines Cat#: ACC737) and cultured in RPMI medium 1640 GlutaMAX (Gibco Cat#: 61870044) according to standard culture protocols. Vero E6 monkey kidney cells (ATCC Cat#: CRL-1586) were kindly provided by Nicola G.A. Abrescia (CIC bioGUNE) and cultured in MEM (Gibco Cat#: 31095-029). HEK 293T cells (Takara Bio Inc. Cat#: 632180) were cultured in DMEM (Gibco Cat#: 41966-029). Media were supplemented with 10% FBS (Thermo Fisher Cat#: 10270106) and 1% Penicillin–Streptomycin (Thermo Fisher Cat#: 15140122). Hypoxia cultures were performed at 1% O2 and 5% CO2 in an In Vivo2 400 hypoxia station (Ruskinn Technologies).
7
Sourcing and Handling Cell Lines
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Human ES cells (H1-line, catalog # WA01) were purchased from the WiCell Research Institute under a material transfer agreement (MTA # 19-W0439). The human iPS cell line (WTC-11) was generously gifted by Dr. Michael E. Ward, National Institute of Neurological Disorders and Stroke (NINDS). The human embryonic kidney (HEK) 293T cells for virus production were commercially available from Takara Bio USA (catalog # 632180).
8
HEK293T and RAW264.7 cell culture and transfection
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HEK293T cells were purchased from TaKaRa Bio (Shiga, Japan), and RAW264.7 cells were a kind gift from Dr. Suda, Showa University, Tokyo, Japan. Cells were maintained in the following media: for HEK293T, minimal essential medium (MEM) (catalog no. 2144225, Nacalai Tesque, Kyoto, Japan) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS); for RAW264.7, MEM supplemented with 10% (v/v) FBS, non-essential amino acid mixture (NEAA, Nacalai Tesque), and L-glutamine (L-GLU, Nacalai Tesque). HEK293T cells were seeded on glass-bottomed dishes (Greiner Bio-one, Kremsmünster, Austria) coated with collagen (Cellmatrix® Type IC; Nitta Gelatin, Osaka, Japan), 2 d before the experiments. The next day, plasmid transfection was performed using Lipofectamine™ 2000 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, and the cells were analyzed 24–36 h after transfection.
9
Keratinocyte Isolation and Lentiviral Transduction
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Primary keratinocytes were isolated from WT CD-1 or TNR mice and cultured using previously described methods (Nowak and Fuchs, 2009 (link)). HEK293T cells were obtained from Takara Bio Inc. and used for lentiviral production as previously described. For lentiviral infections, keratinocytes were plated at 100,000 cells per six wells and incubated with lentivirus in the presence of 100 μl polybrene. 24–48 h after viral infection, cells were selected with 1–2 mg/ml puromycin. 96 h postinfection, keratinocytes were grown to confluency on fibronectin-coated coverslips and shifted to 2 mM Ca2+ growth media as previously described (Ezratty et al., 2011 (link)). Coverslips were fixed in either −20 Methanol (to visualize the microtubule cytoskeleton) or 4% PFA before processing for IF. Paraformaldehyde-fixed samples were permeabilized for 5 min in 0.5% Triton X-100. All antibodies were diluted in 10% normal goat serum in PBS.
10
Culturing HEK 293T, 293T/ACE2 and Expi293F Cells
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HEK 293T cells (#632180, Takara Bio, Kusatsu, Japan) and their derivative, 293T/ACE2 (GenBank accession number NM_001371415) cells, were cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, WAKO) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) and penicillin/streptomycin (100 U/mL, Thermo Fisher Scientific, Waltham, MA, USA). Expi293F cells (#A14527, Thermo Fisher Scientific, Waltham, MA, USA) were grown in Expi293 Expression Medium (Thermo Fisher Scientific, Waltham, MA, USA) containing penicillin/streptomycin (100 U/mL, Thermo Fisher Scientific, Waltham, MA, USA) at 130 rpm, 8% CO2, 37 °C. All cell lines routinely tested negative for mycoplasma contamination.
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