Signaling assays for β2AR-APEX and β2AR wild-type were performed as described previously with some modifications (Liberles and Buck, 2006 (link)). HEK293T (ATCC) cells were seeded in 96-well plates to achieve 70–80% confluency on the day of transfection. Each well was transfected with 20 ng each of a pcDNA5 encoding β2AR-APEX/β2AR wild-type and CRE-SEAP reporter plasmid (BD Biosciences). Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) following manufacturer’s instructions. After incubation of the cells with the transfection mixture and serum-free media for 5 hours at 37 °C , transfec tion mixtures were aspirated and replaced with fresh DMEM with 4.5 g/L glucose and L-glutamine, no pyruvate (VWR), supplemented with 10 µg/mL Gentamicin (VWR). Then transfected cells were treated with indicated final concentrations of isoproterenol (Sigma Aldrich) and incubated at 37 °C for 48 hours followed by 2 hours of incubation a t 70°C. Supernatant from each well was mixed with an equal volume of 0.12 mM 4-methylumbelliferyl phosphate (Sigma Aldrich) in 2 M diethanolamine bicarbonate (diethanolamine from Alfa Aesar diluted in Milli-Q and supplemented with dry ice), pH 10, and incubated for 9 minutes at room temperature. Lastly, the fluorescence was measured using EnVision 2103 Multilabel Reader (PerkinElmer).
Diethanolamine bicarbonate
Manufactured by Merck Group
Diethanolamine bicarbonate is a chemical compound used in various laboratory applications. It is a white crystalline solid that is soluble in water and has a slightly basic pH. The main function of diethanolamine bicarbonate is to serve as a buffer solution, maintaining a stable pH environment in laboratory experiments and processes.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Envision 2103 multilabel reader, by PerkinElmer (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Gentamicin, by Avantor (1 mentions)
4 methylumbelliferyl phosphate, by Merck Group (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
4 methylumbelliferyl phosphate 4 mup, by Merck Group (1 mentions)
2 protocols using diethanolamine bicarbonate
1
Signaling Assay for β2AR Variants
2
SEAP-Based Reporter Assay in HEK293 Cells
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HEK293 cells were plated onto poly-d -lysine (Sigma–Aldrich) preincubated 96-well plates (Corning) with 50 µl growth medium (DMEM medium with 10% BCS and 5% penicillin–streptomycin solution) and cultured for 18–24 h. Receptor plasmids were cotransfected with Cre-SEAP plasmid using polyethylenimine (PEI, Polysciences) and incubated at 37 °C with 5% CO2. About 4 h later, cells were incubated with or without test compounds diluted in serum-free DMEM for 48 h, followed by incubating for 2 h at 70 °C. After returning to room temperature, 50 µl supernatant from each well was incubated with an equal volume of 0.3 mM 4-methylumbelliferyl phosphate (4-MUP, Sigma–Aldrich) in 2 M diethanolamine bicarbonate (Sigma–Aldrich), pH 10.0 for 15 min. Fluorescence was measured with a BioTek Microplate reader.
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