Wild-type rabbit TRPV2 and the truncated TRPV2 construct were expressed in HEK293T cells (ATCC). HEK293T cells were plated on glass coverslips and transiently transfected with a mixture of DNA, Opti-MEM and FuGENE (Promega) according to the manufacturer's manual. Cells were seeded at 60 × 103 per well in the presence of 10 μM ruthenium red and incubated for 48 h at 37 °C. The transfected cells were washed in Hank's buffer and incubated with 2 μM Fura-2 AM in the presence of pluronic acid (Invitrogen) for 30–60 min. Imaging was performed as previously described56 (link). In brief, frames were recorded every second at 340 nm and 380 nm, and the data were analyzed with Nikon Elements software.
Hek293t cells
Manufactured by Promega
Sourced in United States
HEK293T cells are a widely used human embryonic kidney cell line. They are genetically engineered to express the SV40 large T antigen, which allows for increased transfection efficiency and protein expression. HEK293T cells are commonly used in various biological research applications, including protein expression, virus production, and cell-based assays.
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Lab products found in correlation
Hek293t cell, by American Type Culture Collection (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Pluronic acid, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Promega (2 mentions)
Fugene, by Promega (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Metaxpress software, by Molecular Devices (1 mentions)
Hek293t, by GE Healthcare (1 mentions)
Prism 8, by GraphPad (1 mentions)
Glosensor 22f, by Promega (1 mentions)
Psicheck 2, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
14 protocols using hek293t cells
1
TRPV2 Calcium Imaging in HEK293T Cells
2
Transient Transfection of HEK293T Cells
3
NFAT5 3'UTR Luciferase Assay
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HEK293T cells (ATCC) were cultured in 48-well plates for 24 h. pRL-TK luciferase reporter plasmid (Promega, Madison, WI) was used to construct NFAT5 wild type (WT) 3′UTR or mutant (Mut) plasmids, which were then cotransfected with 50 nmol/L miR-31 mimic or mimic-NC into HEK293T cells for 48 h. The Dual Luciferase Reporter Assay System (Promega) was employed to quantify the relative luciferase activity, with Renilla luciferase used as an internal reference.
4
Quantitative HEK293T Cell Imaging Protocol
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HEK293T cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were assessed for contamination using a PCR-based mycoplasma detection kit (Cell-Safe, BioMycoX). HEK293T cells were grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; PAA Laboratories GmbH) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin (Invitrogen). Cells were incubated at 37 °C in a humidified 10% CO2 incubator. For experiments, dissociated HEK293T cells were plated at a density of 1 × 104 cells/well in 96-well plates and transfected in duplicate with 200 ng total DNA using a Fugen6 transfection kit (Promega) according to the manufacturer’s protocol. Cells were stained with 500 ng/ml Hoechst 33342 (Invitrogen) for 30 min at 37 °C before acquiring images. Images were acquired at the indicated time points with an ImageXpress Micro XLS automated epifluorescence microscope (Molecular Devices) using a ×20 Plan Fluor objective and a 4.66 megapixel CMOS camera with a 16-bit readout. Image analysis was performed using MetaXpress software (Molecular Devices).
5
Measuring CCK1R/CCK2R G-protein Signaling
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CCK1R and CCK2R Gs-mediated Gs-cAMP accumulation assays were performed using HEK293T cells (ATCCCRL-11268) transiently expressing human CCK1R and the cAMP biosensor GloSensor-22F (Promega). Cells were seeded (4 × 103 cells, 40 μL/well) into 384-well culture plates and incubated for 24 h at 37 °C in 5% CO2. Next day, the culture medium was removed and the equilibration medium containing 4% (v/v) dilution of the GloSensor™ cAMP reagent stock solution was added to each well. To obtain the concentration-response curves, serially diluted agonists were added to each well to stimulate the cells. The luminance signal was measured using 0.5-s intervals after ligand addition (TECAN, 25 °C). Concentration-responses were generated from the peak response. cAMP accumulation was analyzed by a standard dose-response curve using GraphPad Prism 8.0 (GraphPad Software). EC50 and pEC50 ± SEM were calculated using nonlinear regression (curve fit). Data were means ± SEM from at least three independent experiments performed in technical triplicates. Span, sample size, and expression are provided in Supplementary Tables S2 , S5 , and S6 . Dose-response curves are shown in Supplementary Fig. S8 .
6
NF-κB Transcriptional Activity Assay
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HEK293T cells were purchased from the American Type Culture Collection (Rockville, MD, United States). The cells were cultured in DMEM containing 10% fetal calf serum (FBS), 0.1 mg/mL streptomycin and 100 U/mL penicillin, and grown at 37°C, 5% CO2. Plasmid pGL4.32 (100 ng per well) and Renilla luciferase reporter vector plasmid pRL-TK (9.6 ng per well) were co-transfected into HEK 293T cells using the NF-κB luciferase reporter plasmid pGL4.32 (100 ng per well).
The cells were incubated for 1 day before adding seven commercial anti-inflammatory compounds. The cells were pre-treated with the representative compounds (protocatechuic acid, caffeic acid, ferulic acid; quercetin, luteolin, naringenin, and kaempferol, 10 mg/L) and Dex (4 mg/L) for 1 h and then stimulated with TNF-α (10 ng/mL) for 6 h. After stimulating the cells, HEK 293T cells were lysed and assayed for luciferase activity using a dual luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI, United States).
The cells were incubated for 1 day before adding seven commercial anti-inflammatory compounds. The cells were pre-treated with the representative compounds (protocatechuic acid, caffeic acid, ferulic acid; quercetin, luteolin, naringenin, and kaempferol, 10 mg/L) and Dex (4 mg/L) for 1 h and then stimulated with TNF-α (10 ng/mL) for 6 h. After stimulating the cells, HEK 293T cells were lysed and assayed for luciferase activity using a dual luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI, United States).
7
Luciferase Assay for MLL3 3'UTR
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HEK293T cells (ATCC) were cultured in an incubator at 37 °C with 5% CO2 and saturated humidity, and DMEM (Sigma-Aldrich) was renewed once every 2 or 3 days. The HEK293T cells were co-transfected with wild-type (WT) or mutant type (MUT) MLL3 3′-untranslated region (3′-UTR) psiCHECK-2 plasmid (Promega) and miR-130b-3p/mimic or mimic NC using lipofectamine 2000 (Invitrogen). Cell lysates were collected 48 h post transfection and then firefly and renilla luciferase activities were measured by a dual luciferase reporter assay kit (Promega) based on the manufacturer’s protocol. Renilla luciferase activity was utilized for normalization.
8
Generation of ARE/mVP24 and ARE/GFP HEK293T Cell Lines
9
TRPV2 Calcium Imaging in HEK293T Cells
10
Optimized Recombinant Protein Expression
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For protein expression of selected TGM family members, the signal peptide sequence was determined (using the online server SignalP 4.1) and the resulting mature sequence of each (i.e. without signal peptide) was synthesized by GeneArt (Thermo Fisher Scientific, UK) codon-optimised with flanking AscI/NotI restriction digest sites and inserted into a holding vector; codon-optimised nucleotide sequences are presented in Supplementary Table S2 . Each family member DNA was subsequently subcloned into mammalian expression vector pSECTag2A (Invitrogen, USA) using restriction sites AscI and NotI, and resultant constructs were sequence verified.
Ten micrograms of purified plasmid DNA of each construct was transiently transfected into HEK293T cells using the calcium chloride method (Promega, USA). Cells were switched into serum-free media (293 SFM II; Gibco, USA) after 24 h, and cultured for up to 4 days, after which point supernatants were collected, filtered through a 0.45 µM filter and dialysed into imidazole-free binding buffer before being loaded onto a 1 ml nickel sulphate charged HiTrap Chelating HP column (GE Healthcare, USA) using a peristaltic pump. Proteins were purified using an Akta Purifier (GE Healthcare) and positive fractions were dialysed into PBS.
Ten micrograms of purified plasmid DNA of each construct was transiently transfected into HEK293T cells using the calcium chloride method (Promega, USA). Cells were switched into serum-free media (293 SFM II; Gibco, USA) after 24 h, and cultured for up to 4 days, after which point supernatants were collected, filtered through a 0.45 µM filter and dialysed into imidazole-free binding buffer before being loaded onto a 1 ml nickel sulphate charged HiTrap Chelating HP column (GE Healthcare, USA) using a peristaltic pump. Proteins were purified using an Akta Purifier (GE Healthcare) and positive fractions were dialysed into PBS.
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