HEK293 cells (ATCC CRL-1573.3) were cultured in growth media (DMEM, 10% heat inactivated fetal bovine serum, penicillin (100units/mL), streptomycin (100 μg/mL) and L-glutamine (290 μg/mL)) and used between passages 5–25. For GPCR heterologous expression and functional assays, cells were transfected (Lipofectamine, 2000) at 80% confluency approximately 16 h after seeding on T-25 culture flasks with a 1:1 ratio of Sm.5HTRL and the pGloSensor 22-F plasmid (Promega). Sm.5HTRL was codon optimized for human expression and subcloned into the pcDNA3.1(−) mammalian expression vector. Sm.5HTRL (GenBank accession, KX150867) is a longer form of the Sm.5HTR originally reported by Patocka et al. (Patocka et al., 2014 (link)). The following day, cells were trypsinized, centrifuged (300 g/5 min), resuspended in DMEM supplemented with 1% dialyzed FBS (Gibco) and plated in 96 well, solid white plates (Corning, cat # 3917). After overnight culture to allow adherence, media was exchanged for assay buffer (HBSS supplemented with 0.1% BSA, 20 mM HEPES (pH 7.4), and GloSensor reagent (Promega)). cAMP-luminescence assays were performed in the presence of phosphodiesterase inhibitor (IBMX, 200 μM) using a GloMax®-Multi Detection System plate reader (Promega).
Pglosensor 22 f plasmid
Manufactured by Promega
Sourced in United States
The PGloSensor 22-F plasmid is a laboratory tool used for the detection and measurement of gene expression. It contains a synthetic luciferase reporter gene that can be used to quantify transcriptional activity in various cell systems.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glosensor reagent, by Promega (2 mentions)
Glomax multi detection system plate reader, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
6 protocols using pglosensor 22 f plasmid
1
Heterologous Expression of Sm.5HTR_L in HEK293 Cells
2
Heterologous GPCR Expression in HEK-293 Cells
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HEK-293 cells (ATCC CRL-1573.3) were cultured in growth media [DMEM (Gibco), 10% heat inactivated fetal bovine serum (Gibco), penicillin (100units/mL), streptomycin (100μg/mL) and L-glutamine (290μg/mL)] and used for assays between passages 5 and 25. For cestode GPCR heterologous expression assays, cells were transfected (Lipofectamine 2000, Invitrogen) at 80% confluency approximately 16 hours after seeding within T-25 culture flasks with a 1:1 ratio of human codon optimized cestode GPCR cDNA (subcloned into a pcDNA3.1(-) mammalian expression vector) and cDNA encoding the pGloSensor 22-F plasmid (Promega). The following day, cells were trypsinized, centrifuged (300g/5min), resuspended in DMEM supplemented with 1% dialyzed FBS (Gibco) and plated in 96 well, solid white plates (Corning, cat # 3917). After overnight culture to allow adherence, media was exchanged for assay buffer (HBSS supplemented with 0.1% BSA, 20mM HEPES (pH 7.4), and GloSensor reagent (Promega). cAMP-luminescence assays were performed following addition of a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX; 200μM) using a GloMax-Multi Detection System plate reader (Promega) as described previously [24 (link)].
3
GloSensor cAMP Assay in PC12 Cells
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The GloSensor cAMP assay was conducted with the same parameters (50,000 PC12 cells per 96-well) as described above for the firefly luciferase assay, with however, three differences. Specifically, (1) 30 ng/well of the pGloSensor-22F plasmid (Promega, E2301) was used for transfection, and (2) on DIV3, cells were equilibrated in assay medium containing a 2% v/v dilution of D-luciferin (Synchem, bc219) for 2 h at 37°C before (3) the treatment of compounds for 15 min and lysis in 1x passive lysis buffer (Promega) and luciferase activity measurement.
4
cAMP Assay in CHO-hCB2 Cells
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cAMP assays were performed in CHO-hCB2 stably transfected with the pGloSensorTM 22-F plasmid (Promega, Madison, WI, USA) as previously described [22 (link)]. Briefly, cells were seeded in 96-well plate and kept in an equilibration medium (GloSensor™ cAMP reagent diluted in 5% of a CO2-independent medium plus 10% FBS) for 2 h at RT in the dark. Compounds or vehicle were diluted in an assay medium containing 1 μM of forskolin and 250 μM of 3-isobutyl-1-methylxanthine (IBMX). Chemiluminescence was recorded after 20 min. Non-specific signal was determined in cells with only IBMX. Experiments were performed in the presence and after removal of the constitutive activity of the receptors following the same procedure described above. Results were normalized by subtracting the residual cAMP production and expressed amount of cAMP formed.
5
cAMP Assay in Engineered CHO Cells
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cAMP assays were performed in CHO-K1-hCB2 stably transfected with the pGloSensorTM 22-F plasmid (Promega) as previously described48 (link). Briefly, cells were seeded in 96-well plate and after attachment the medium was replaced by 40 µL of equilibration medium (GloSensor™ cAMP Reagent diluted in 5% v v−1 of CO2-independent medium plus 10% FBS). The plates were incubated for 2 h at RT in the dark. Compounds or vehicle were diluted in 10 µL assay medium containing 1 µM of forskolin and 250 µM of 3-isobutyl-1-methylxanthine (IBMX). Chemiluminescence was monitored continuously starting from immediately after the addition and for 30 min (keeping the plate at RT and in the dark). Basal levels of chemiluminescence (cells plus IBMX and vehicle without forskolin) were subtracted to the measured values. Results were normalized by subtracting the residual cAMP production and expressed as % of vehicle control.
6
Somatostatin Receptor Mediated cAMP Assay
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HEK-293T cells (CRL-3216 from ATCC, procured from NCCS, Pune) were co-transfected with human somatostatin receptor subtypes 2 & 5, plasmid cDNA, and cAMP biosensor (pGloSensorTM-22F plasmid; Promega Corp.) using PEImax. Ligand incubation was followed by the addition and incubation of forskolin (FSK) to the plate. Dose-dependent inhibition of forskolin-induced cAMP production by the peptide ligands was measured using a luminescence plate reader. An elaborate description of the assay setup has been provided in Supplementary Information.
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