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Hek293 cells

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HEK293 cells are a widely used human cell line derived from human embryonic kidney cells. These cells are commonly used in cell biology research and biotechnology applications due to their ability to efficiently express recombinant proteins. HEK293 cells provide a reliable and well-established model system for various experimental purposes.

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18 protocols using hek293 cells

1

Interferon-γ Stimulation Assay in HEK293 Cells

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One million HEK293 cells (Promega) were plated on a T25 dish. After 8 h, cells were transfected with 5 μg of a GAS reporter vector (pGL4[luc2P/GAS-RE/Hygro], Promega) and 15 μl FuGENE HD (Promega, cat no. E2311) according to the manufacturer’s instructions. 16 hours post-transfection, cells were plated on pre-plated siRNAs as described above. Cells were incubated on siRNAs for 48 hours before activation with 100 ng/ml IFNγ (Peprotech) after which incubation was continued for 24 hours. Subsequently, cell viability and reporter activity were measured with CellTiter-Fluor and ONE-Glo reagents (Promega), respectively.
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2

Transient and Stable Transfection Assays

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HEK293 cells (ATCC) were transfected with MSCV-N-GFP, MSCV-N-6E6, MSCV-N-11E6, and MSCV-N-16E6 plasmids using Fugene 6 (Promega). Forty-eight hours post transfection, cells were lysed and used for co-immunoprecipitation assay. For stable transfection, HEK293 cells were transfected with the same amount of MSCV-N-GFP and MSCV-N-E6 constructs using Fugene 6 (Promega) and selected with puromycin for 2 weeks. Then single clones were then isolated and propagated for co-immunoprecipitation assays.
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3

Cloning and Mutagenesis of Hepatic Transcription Factors

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The PCR primers were synthesized by Integrated DNA Technologies (IDT). Total genomic DNA extracted from liver mouse and HEK293 cells (ATCC) were used as templates for PCR-cloning of the mouse/human promoter/intron fragments into the KpnI/MluI sites of the pGL3 basic luciferase reporter vector (Promega). The glutamic acid (E) to lysine (K) at 363 (E363K) mutant of pCDNA3-HNF4A2 was generated using Q5® Site-Directed Mutagenesis Kit (New England Biolabs). The pcDNA3- small heterodimer partner (SHP)-Flag vector for SHP was synthesized by GenScript. The primers and sequence information of all reporter constructs is provided in Supplemental Table S1 (https://figshare.com/s/650749d0f09c1c5d4b2e). All the constructed vectors were verified by sequencing.
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4

Quantifying SEAP Reporter Gene Activity

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HEK293 cells (obtained from National Infrastructure of Cell Line Resource, Beijing, China) were seeded in a 96-well plate at a density of 5,000 cells per well and transfected with the SMAD-binding element (SBE)-SEAP plasmid using Fugene HD transfection reagent (Promega Corp.), as described previously (19 (link)), and the pSEAP basic plasmid as a negative control (both from Clontech Laboratories, Inc., Mountainview, CA, USA) prior to treatment with TGF-β1 and baicalin/baicalein. The SEAP signal was quantified using the SensoLyte® p-nitrophenyl phosphate (pNPP) SEAP Reporter Gene assay kit (AnaSpec, Fremont, CA, USA) according to the manufacturer's protocol. Briefly, after treatment with baicalin/baicalein (20–80 µM) for 48 h, cell supernatant was collected and heated at 65°C for 30 min to inactivate non-specific alkaline phosphatase activity. The assay buffer was then added and the mixture was incubated for 5 min at room temperature. Subsequently, pNPP substrate buffer was added and the mixture was incubated for 1 h at room temperature. OD values were then measured at 405 nm.
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5

Quantifying VEGF-induced NFAT Activation

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KDR/NFAT-RE HEK293 cells (Promega, Cat#:GA2001) were engineered to express Luc2P under the control of the NFAT response element, as well as exogenous KDRs. Assays were performed according to the manufacturer's instructions. Briefly, KDR/NFAT-RE cells were seeded at 4 × 104 cells/well in 50 μl in white 96-well plates. Then, 25 μl of 3× serially diluted antibodies in DMEM containing 10% FBS and 30 ng/ml VEGF was added to the plates and incubated in a 37 °C humidified incubator with 5% CO2 for 6 h. Then, 75 μl of Bio-Glo™ Reagent was added to each well, and the plates were incubated at room temperature for 5–30 min. Luminescence was measured on a SpectraMax i3x.
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6

Transient Transfection of HEK293 Cells

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HEK293 cells (ATCC, Manassas, VA, USA) were cultured in MEM with 10% FBS at 37°C and 5% CO2. For transient transfections, cells were transfected with plasmids described above by Xtremegene (Roche, Indianapolis, IN, USA) and used two days post-transfection. For the cAMP pGlo and ELISA assays, cells were transfected in one batch and then split for use in each assay. HEK293 cells stably expressing pGloSensor-20F cAMP plasmid (Promega) under Hygromycin selection were transfected with HA-MC1R, or HA-MC1R harboring one of the variants, in wells of a 6-well dish. One day post-transfection, approximately 10,000 cells per well were added to white bottom (cAMP pGlo Assay) or clear poly-L-lysine coated (ELISA) 96 well dishes.
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7

Transfection and Proteasome Inhibition of KCNQ1

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HEK293 cells were purchased from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere with 5% CO2. HEK293 cells were plated into six-well plates and transfected with 0.5 μg WT or mutant myc-KCNQ1 per well at 30 to 50% confluence using FuGENE 6 transfection reagent (Promega). When WT and mutant myc-KCNQ1 were cotransfected, the WT/mutant or WT/pIRES2-EGFP vector ratio was 1:1 and total DNA was 0.5 μg. Approximately 48 hours later, cells were prepared for flow cytometry measurements. To assess the effect of proteasome inhibitor on myc-KCNQ1 expression, we treated cells starting 1 day after transfection with 25 μM MG132 or 0.1% dimethyl sulfoxide (DMSO) (control) for 20 hours before quantitating KCNQ1 protein levels.
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8

Stable Cell Lines for GPCR Studies

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HEK293 cells (American Type Culture Collections) stably expressing KORGFP, mycKOR, mycMOR, HA-D2DR, mycKOR and HA-D2DR, mycKOR and HA-PRDX6, or mycKOR (puro) and FLAG-Gαi3 were generated by transfecting HEK293 cells using Fugene HD (Promega) according to manufacturer instructions. HEK293 cells were maintained in Dulbecco’s modified medium/F12 (Fisher Scientific) with 10% fetal bovine serum (Sigma-Aldrich) and penicillin-streptomycin-L-glutamine (Fisher Scientific). HEK293 cells expressing mycKOR or mycMOR were grown in media supplemented with G418 (200 µg ml−1) (Fisher Scientific); HEK293 cells expressing HA-D2DR were grown in media supplemented with hygromycin (50 µg ml−1) (Fisher Scientific); HEK293 cells expressing mycKOR and HA-D2DR or HA-PRDX6 were grown in media supplemented with G418 (200 µg ml−1) and hygromycin (50 µg ml−1); HEK293 cells expressing mycKOR (puro) and FLAG-GNAI3 were grown in media supplemented with puromycin (0.5 µg ml−1) (Sigma-Aldrich) and G418 (200 µg ml−1). For SILAC cell culture, cells were grown for five doublings in 15 cm plates in custom DMEM media (Caisson Labs) containing isotope-labeled lysine and arginine (light media or L:R0K0 = lys0/arg0; medium media or M:R6K4 = lys4/arg6; heavy media or H:K10R8 = lys8/arg10) (Cambridge Isotope Labs).
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9

Quantitative Analysis of ADAM10 Expression

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The hAdam10 D + C domains (AA454–673) were amplified by PCR and cloned into the pDisplay vector (Thermofisher, Waltham, MA, USA) using BglII and SalI restriction enzyme sites (retaining the N terminal HA tag and Myc at the membrane). The construct was transfected into HEK293 cells (Fugene, Promega), and a stable line was generated through antibiotic (neomycin) selection.
For quantitative PCR, cDNA generated from RNA extracts (QIAGEN RNeasy) of snap-frozen tumor samples was analysed using an iTaq Universal SYBR Green kit (Bio-Rad Laboratories, Hercules, CA, USA) and a RotorGene 3000 cycler (Corbett Research). Primers specific for human ADAM10 were used to determine expression by comparative CT (ΔΔCT), relative to the averaged expression of three housekeeping genes (GAPDH, Beta 2 microglobulin, and Eukaryotic translation initiation factor 4E).
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10

Gs22a Cells for cAMP Reporting

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Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.)28 (link). The cells were cultured in DMEM supplemented with fetal bovine serum (10%) in a humidified incubator containing 5% CO2 and set at 37 °C, and seeded into 96- or six-well plates for transient DNA transfection and functional assays. The cells were transfected when the monolayers were 85–95% of confluency using Lipofectamine™ 2000 (ThermoFisher, Cat. No 11668019) and 100 ng of DNA per well for 96-well plates and 1000 ng of DNA per well for 6-well plates, except for DNA-titration studies, in which 150 ng of total DNA was used for 96-well plates (plate reader assays) and 2000 ng of total DNA was used for six-well plates (flow cytometry assays), with the total amounts of DNA being comprised of a quantity of receptor-expressing plasmid DNA ranging from 2 ng/well to 150 ng/well for 96-well plates, and from 60 ng/well to 2000 ng/well for six-well plates, and an appropriate quantity of pCDNA1 vector DNA such that the total amount of DNA was equal in all wells. Transfection mixtures were prepared in Opti-Mem™ (Gibco, Cat. No 31985070) and contained 3 μl of Lipofectamine™ 2000 per μg of DNA. Assays were performed 48 hours after transfection, and media was changed 2–4 h prior to assay.
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