pcDNA4TO-TS-SNAP-β2AR or pcDNA4TO-TS-SNAP-A2A were stably transfected into T-RexTM-293 cells (Invitrogen) using polyethylenimine (PEI). A mixed population stable line was selected by resistance to 5 μg/mL blasticidin and 20 μg/mL Zeocin. Stable cell lines were maintained in high glucose DMEM (Sigma D6429) with 10% foetal bovine serum (FBS), 5μg/μL blasticidin and 20μg/μL zeocin, at 37°C and 5% CO2. When ∼70% confluent TS-SNAP-β2AR expression was induced with 1μg/mL tetracycline. Cells were left to express for 50hrs before harvesting.
T rextm 293 cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The T-RexTM-293 cells are a stable, tetracycline-inducible mammalian cell line derived from the human embryonic kidney 293 cell line. The cells are designed to express a gene of interest under the control of a tetracycline-regulated promoter system.
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Lab products found in correlation
Dmem high glucose, by Merck Group (1 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (1 mentions)
L glutamine, by Sartorius (1 mentions)
Blasticidin ant bl 1, by InvivoGen (1 mentions)
Mc mir 2300, by Mirus Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Culture vessels, by Corning (1 mentions)
Flp frt system, by Thermo Fisher Scientific (1 mentions)
Antibodies against strepii tag, by Novus Biologicals (1 mentions)
4 protocols using t rextm 293 cells
1
Stable Expression of GPCR Constructs
2
Stable Expression of SNAP-Tagged GPCRs
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The construct pcDNA4TO-TwinStrep(TS)-SNAP-β2AR was generated by amplification of the SNAP and β2AR sequences of the pSNAPf-ADRB2 plasmid (NEB) and insertion into pcDNA4TO-TS using Gibson assembly (Heydenreich, Miljus et al. 2017) . The construct pcDNA4TO-TS-SNAP-A2A was generated by amplifying the A2A receptor from the pDNA3.1 SNAP A2A construct described in (Comeo, Kindon et al. 2020 ) and inserting into pcDNA4TO-TS-SNAP vector using Gibson assembly. This therefore gave the construct pcDNA4TO-TS-SNAP-A2A. Both constructs used a signal peptide based on the 5HT3A receptor to increase protein folding and expression.
Transfection and mammalian cell culture pcDNA4TO-TS-SNAP-β2AR or pcDNA4TO-TS-SNAP-A2A were stably transfected into T-Rex TM -293 cells (Invitrogen) using polyethylenimine (PEI). A mixed population stable line was selected by resistance to 5 µg/mL blasticidin and 20 µg/mL Zeocin. Stable cell lines were maintained in high glucose DMEM (Sigma D6429) with 10% foetal bovine serum (FBS), 5μg/μL blasticidin and 20μg/μL zeocin, at 37°C and 5% CO2. When ~70% confluent TS-SNAP-β2AR expression was induced with 1μg/mL tetracycline. Cells were left to express for 50hrs before harvesting.
Transfection and mammalian cell culture pcDNA4TO-TS-SNAP-β2AR or pcDNA4TO-TS-SNAP-A2A were stably transfected into T-Rex TM -293 cells (Invitrogen) using polyethylenimine (PEI). A mixed population stable line was selected by resistance to 5 µg/mL blasticidin and 20 µg/mL Zeocin. Stable cell lines were maintained in high glucose DMEM (Sigma D6429) with 10% foetal bovine serum (FBS), 5μg/μL blasticidin and 20μg/μL zeocin, at 37°C and 5% CO2. When ~70% confluent TS-SNAP-β2AR expression was induced with 1μg/mL tetracycline. Cells were left to express for 50hrs before harvesting.
3
Tetracycline-Inducible T-REx-293 Cell Culture
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T-RExTM-293 cells (Thermo-Scientific, Waltham, MA, USA) were grown at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, 01-055-1A, Biological Industries, Beit-Haemek, Israel) supplemented with 10% TET-System approved fetal bovine serum (04-005-1A, Biological Industries), 1% Penicillin-Streptomycin (03-031-1B, Biological Industries), 2 mM L-glutamine (03-020-1B, Biological Industries) and 5 mg/mL Blasticidin (ant-bl-1, InvivoGen, San Diego, CA, USA). Cells were not used above passage 30. Transfections were performed using TransIT-LT1 (MC-MIR-2300, Mirus Bio, Madison, WI, USA) transfection reagents, according to the manufacturer’s protocol. Tetracycline was added 18–24 h prior to experiments to achieve maximum saturating levels of plasmid expression.
4
Engineered Flp-In T-REx 293 Cell Lines
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Flp-InTM T-RExTM293 cells (Thermo Fisher, Cat#R78007) harboring IC2C wt or ∆ICN with C-terminal SNAP-FLAG-strepII tagwhich were generated using the FLP/FRT system (Thermo Fisher). Briefly, pcDNA5-FRT-TO construct and pOG44, which expresses Flipase, were co-transfected using Lipofectamine 2000 (Thermo Fisher) into Flp-InTM T-RExTM293 cells. After recovery from transfection, cells were grown in DMEM containing 10% FBS, 1% Penicillin-Streptomycin, and 100 µg/mL Hygromycin B to select cells in which pcDNA5 construct was inserted into FRT locus. The expression of tagged IC2C was verified by western blotting with antibodies against StrepII tag (Novus Biologicals). These cell lines were grown in culture vessels (Corning, Fisher Scientific) to 80% confluence, and then tagged IC2C expression was induced with 1 µg/ml doxycycline for two days. Cells were harvested in PBS by tapping the culture vessels, and spun down at 1200 rpm for 5 min in Sorvall Legend XTR. Cell pellets were washed with PBS, snap frozen in liquid nitrogen, and stored at −80 °C.
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