293a cells

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293A cells are a type of human embryonic kidney cell line that are commonly used in cell biology research and the production of recombinant proteins. They are derived from human embryonic kidney cells and have been immortalized through the introduction of the adenovirus E1A and E1B genes. 293A cells have the ability to be easily transfected and can be used to produce high levels of recombinant proteins.

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HEK293A (293A) cell line 293A cell line

45 protocols using 293a cells

Generating Adenoviral Vectors Expressing shRNA and EGFP

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Vectors expressing shRNA targeting rhesus PGR mRNA (shPGR), scrambled shRNA (shScram), or the gene encoding enhanced GFP (EGFP) via cytomegalovirus promotor (CMV) were constructed using the ViraPower system (Life Technologies). Briefly, the nucleotide expression cassette (encoding either shRNA or EGFP) was transferred from the pENTR/U6 entry plasmid to the adenoviral destination plasmid (pAd/BLOCK-iT) via Gateway recombination. The resulting adenoviral plasmids were then transfected into 293A cells (Life Technologies) to generate recombinant vectors. For production, viruses were expanded and harvested from cells by triple freeze-thaw method. Cell lysates were treated with Benzonase nuclease (Sigma-Aldrich) to digest genomic DNA and virions were purified by iodixanol gradient ultracentrifugation [14 (link)] using OptiPrep medium (Sigma-Aldrich). The 25%-40% iodixanol interfaces containing adenovirus were aspirated and buffer exchanged into PBS + 5% glycerol using Amicon Ultra-15 Centrifugal Filter Units (EMD Millipore). To assess biological activity, virus preparations were titered by TCID50 assay on 293A cells, and values were converted to plaque-forming units (pfu). The absence of replication-competent adenovirus was verified using an agarose gel overlay method on A549 indicator cells [15 (link)].

Bishop C.V., Hennebold J.D., Kahl C.A, & Stouffer R.L. (2016). Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production. Biology of Reproduction, 94(5), 109.

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Adenovirus-Mediated Overexpression of miR-182

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Control adenovirus and adenovirus expressing human SREBP-2 were purchased (Eton Biosciences) and amplified in 293A cells (Life Technologies). Adenovirus expressing the precursor form of mmu-miR-182 was generated using the AdEasy XL Adenoviral Vector System. The DNA sequence from 157-bp upstream through 224-bp downstream of the mouse pri-miR-182 sequence was amplified by PCR with primers containing NheI and XhoI restriction sites on either end. This fragment was cloned into the pGEM-T vector (Promega), sequenced, transferred into the pShuttle vector and subsequently cloned into the pAd-Easy-1 vector (Agilent).

Miao J., Ling A.V., Manthena P.V., Gearing M.E., Graham M.J., Crooke R.M., Croce K.J., Esquejo R.M., Clish C.B., Vicent D, & Biddinger S.B. (2015). Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis. Nature Communications, 6, 6498.

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Adenoviral Vector Construction for miR-182

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Control adenovirus and adenovirus expressing human SREBP-2 were purchased (Eton Biosciences) and amplified in 293A cells (Life Technologies). Adenovirus expressing the precursor form of mmu-miR-182 was generated using the AdEasy^™ XL Adenoviral Vector System. The DNA sequence from 157-bp upstream through 224-bp downstream of the mouse pri-miR-182 sequence was amplified by PCR with primers containing NheI and XhoI restriction sites on either end. This fragment was cloned into the pGEM-T vector (Promega), sequenced, transferred into the pShuttle vector and subsequently cloned into the pAd-Easy-1 vector (Agilent).

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Adenoviral Knockdown of Frizzled Receptors

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All shRNAs were purchased from Sigma (MISSION shRNA library). The KD efficiency was validated as described in Extended Data Fig. 8c, d. ShRNA sequences showing the highest efficiency were selected to generate adenoviruses. Adenoviruses expressing a control shRNA (5’-CTGGACTTCCAGAAGAACA-3’), shRNAs against mouse FZD1 (shRNA#2: 5’-TGGTGTGCAACGACAAGTTTG-3’), or FZD2 (shRNA#5: 5’-CGCTTCTCAGAGGACGGTTAT-3’) were constructed using the Block-it U6 adenoviral RNAi system (Life Technologies), followed by viral packaging and multiple rounds of amplification in 293A cells (Life Technologies).

Tao L., Zhang J., Meraner P., Tovaglieri A., Wu X., Gerhard R., Zhang X., Stallcup W.B., Miao J., He X., Hurdle J.G., Breault D.T., Brass A.L, & Dong M. (2016). Frizzled are colonic epithelial receptors for Clostridium difficile toxin B. Nature, 538(7625), 350-355.

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Overexpression of ABCG2 and URAT1 in 293A Cells

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Human embryonic kidney 293 cell-derived 293A cells were purchased from Life Technologies (Carlsbad, CA, USA) and cultured in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), 1% penicillin/streptomycin, 2 mM L-glutamine (Nacalai Tesque), and 1 × Non-Essential Amino Acid (Life Technologies) at 37°C in an atmosphere of 5% CO₂ as described previously (Toyoda et al., 2016a (link)). All experiments were carried out with 293A cells at passages 10–20. To express human ABCG2 (NM_004827) fused with Myc-tag at its N-terminus (Myc-ABCG2) and EGFP (control), we used Myc-ABCG2 and EGFP-expressing adenoviruses constructed in our previous study (Ito et al., 2015 (link)), respectively. To express the URAT1, open reading frame of URAT1 (NM_144585.3) was cloned into a pcDNA3.1(+) vector (Life Technologies) with a FLAG tag at its N-terminus. To express mouse Abcg2 and EGFP (control), open reading frames of mouse Abcg2 (NM_011920) and EGFP were inserted into a pcDNA3.3 vector (Life Technologies), respectively.

Miyata H., Takada T., Toyoda Y., Matsuo H., Ichida K, & Suzuki H. (2016). Identification of Febuxostat as a New Strong ABCG2 Inhibitor: Potential Applications and Risks in Clinical Situations. Frontiers in Pharmacology, 7, 518.

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Adenoviral Knockdown of Frizzled Receptors

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Transient Transfection of TRPV1 in 293A Cells

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293A cells (Life Technologies Corp.) were cultured in D-MEM medium containing 2 mM L-glutamine and 10% FBS. Cells were seeded at a density of 1 x 10⁴ cells/well in poly-D-lysine-coated 96-well plates (BD Falcon, Franklin Lakes, NJ). After ~24 h of culture at 37°C, transient transfection with human TRPV1 or TRPV1 mutants was performed by using FuGENE HD Transfection Reagent (Promega, Madison, WI) in accordance with the manufacturer’s instructions. After transfection, cells were cultured for 40–48 h in a CO₂ incubator and used for the Ca²⁺ flux assay.

Ohbuchi K., Mori Y., Ogawa K., Warabi E., Yamamoto M, & Hirokawa T. (2016). Detailed Analysis of the Binding Mode of Vanilloids to Transient Receptor Potential Vanilloid Type I (TRPV1) by a Mutational and Computational Study. PLoS ONE, 11(9), e0162543.

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Adenoviral Induction of Pancreatic Transcription Factors

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Recombinant adenoviruses expressing mouse Pdx1, Neurod (kindly gifted by Dr. S. Yoshida), Mafa, and Mafb were prepared using the ViraPower Adenoviral Gateway Expression kit (Life Technologies, Carlsbad, CA, USA). Each cDNA fragment was cloned into a pENTR4 entry vector. To create expression clones and produce recombinant adenoviruses, the pENTR4 inserts were transferred into pAd/CMV/V5-DEST destination vectors using the LR recombination reaction, and PacI-linearized plasmids were transfected into 293A cells (Life Technologies) by using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland). The viruses were propagated in 293A cells and purified using CsCl₂ banding followed by dialysis against 10 mM Tris-HCl, pH 8.0 with 2 mM MgCl. The infectious titers were determined using the Adeno-X Rapid Titer kit (Takara, Shiga, Japan). The successful adenoviral gene transfer is shown in Figure S1A, in which 5×10⁹ infectious units of the viruses were injected into wild-type mice, and immunohistochemistry using the target antibodies was performed 3 days after the infection. 39.6±3.9%, 26.4±5.8%, 46.5±3.2%, 57.3±5.8%, of liver cells reacted to PDX1, NEUROD, MAFA, and MAFB antibodies, respectively (n = 3) (Figure S1A).

Nagasaki H., Katsumata T., Oishi H., Tai P.H., Sekiguchi Y., Koshida R., Jung Y., Kudo T, & Takahashi S. (2014). Generation of Insulin-Producing Cells from the Mouse Liver Using β Cell-Related Gene Transfer Including Mafa and Mafb. PLoS ONE, 9(11), e113022.

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Transfection of ABCG2 Variants in 293A Cells

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Human embryonic kidney 293 cell-derived 293A cells were purchased from Life Technologies (Carlsbad, CA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies), 1% penicillin/streptomycin, 2 mM L-glutamine (Nacalai Tesque), and 1 × Non-Essential Amino Acid (Life Technologies) at 37 °C in an atmosphere of 5% CO₂. Each vector plasmid for ABCG2 WT, p.M131I, or p.R236X was transfected into 293A cells by using polyethyleneimine MAX (PEI-MAX; 1 mg/mL in milli-Q water, pH 7.0; Polysciences, Warrington, PA, USA) as described previously [57 (link)]. The amount of plasmid DNA used for transfection was adjusted per sample group.

Toyoda Y., Pavelcová K., Bohatá J., Ješina P., Kubota Y., Suzuki H., Takada T, & Stiburkova B. (2021). Identification of Two Dysfunctional Variants in the ABCG2 Urate Transporter Associated with Pediatric-Onset of Familial Hyperuricemia and Early-Onset Gout. International Journal of Molecular Sciences, 22(4), 1935.

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Cell Culture Protocol for Vero, FaDu, and 293-A Cells

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Vero cells and FaDu cells (human epithelial cells derived from a squamous cell carcinoma of the pharynx (ATCC, Manassas, VA, USA)) were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% fetal calf serum (growth media). 293-A cells (Thermo Fisher Scientific, Waltham, MA, USA) were maintained in growth media supplemented with 0.1 mM MEM Non-Essential Amino Acids (NEAA) (Thermo Fisher Scientific). BHK-BSR-T7/5 cells [16 (link),35 (link)], kindly provided by K. Conzelmann, (Munich, Germany), were maintained in growth media and 1 mg/mL neomycin.

Malik T., Ngo L., Bosma T, & Rubin S. (2019). A Single Point Mutation in the Mumps V Protein Alters Targeting of the Cellular STAT Pathways Resulting in Virus Attenuation. Viruses, 11(11), 1016.

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