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Pglosensor 20f camp plasmid

Manufactured by Promega

The PGloSensor-20F cAMP plasmid is a laboratory tool used to measure changes in intracellular cyclic AMP (cAMP) levels. It contains a cAMP-responsive element coupled to a firefly luciferase reporter gene, allowing for real-time monitoring of cAMP signaling dynamics in living cells.

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5 protocols using pglosensor 20f camp plasmid

1

Gαi1 regulation of cAMP signaling

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A293 cells (4 × 104 cells/well) were plated per well in a 96-well plate (655983, Greiner). The following amounts of DNA were used per well: 50 ng of pGloSensor™-20F cAMP Plasmid (E1171, Promega), 125 ng of untagged Gαi1-WT, Gαi1-QL or BioID2 fused Gαi clones or empty vector (control, pCDNA3.1+). Reverse transfection was performed using 1:3 DNA: Lipofectamine 2000 ratio. 24 hr after transfection, cells were washed once with 1× PBS, and 75 μL of 2 mM D-luciferin (LUCK-1G, Goldbio) in Leibovitz’s L-15 medium (21083-027, Gibco) was added for 2 hr at 37 °C, 5% CO2 incubator. Cells were treated with vehicle or 1 μM forskolin (Fsk) (11018, Cayman Chemicals) and luminescence was measured using a Varioskan™ LUX multimode microplate reader for 30 min.
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2

Measuring cAMP and Ca2+ in Rhodopsin-HEK293S Cells

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The intracellular cAMP and Ca2+ levels in rhodopsin-expressing HEK293S cells (human embryonic kidney 293 S cells, provided by Dr. Jeremy Nathans of Johns Hopkins University) were measured using the GloSensor cAMP assay and the aequorin assay, respectively, as described previously (Bailes and Lucas, 2013 (link)). HEK293S cells have been confirmed to be free from mycoplasma contamination. The identity of HEK293S cells was confirmed by similarity to HEK293 and HEK293T cells through STR profiling, and by morphological observation of the cells. The pGloSensor-20F cAMP plasmid (Promega) was used for the GloSensor cAMP assay. The wild type aequorin obtained by introducing two reverse mutations into the plasmid [pcDNA3.1+/mit-2mutAEQ] (Addgene #45539) (de la Fuente et al., 2012 (link)) was used for the aequorin assay. The rhodopsin expression plasmids were constructed based on pCS2+ (see the Zebrafish section) and used for transfection. For Gαq inhibition, YM-254890 (FUJIFILM Wako Pure Chemical Corp., 257–00631, Osaka, Japan) was added (1 μM) 5 min before the measurement. Green (500 nm) and violet (410 nm) LED lights were applied for 5 s in the GloSensor cAMP assay and for 1 s in the aequorin assay as light stimuli. Dual Head LED Light 505 nm (GB Life Science) and SPL-25-CC (REVOX, Inc) were used for green and violet LED light stimulation, respectively.
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3

Quantifying cAMP in HEK293 Cells

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HEK293 cells stably expressing pGloSensor-20F cAMP plasmid (Promega) under Hygromycin selection were transfected with mCherry-2A-MC4R or the truncation mutants in wells of a 6 well dish. The next day cells were re-plated in 12 wells of a white bottom 96 well dish (for cAMP assay) and wells of a clear bottom plate (to determine expression). The following day, the media was removed from the clear bottom dish and replaced with Imaging buffer and the fluorescence was read on a Spectramax M3 plate reader (Molecular Devices). Fluorescence from empty-vector transfected cells was subtracted from each group and then the values were normalized to the fluorescence reading for the wild-type mCherry-2A-MC4R from each experiment. Data is from 3 independent experiments (mean ± SEM). For the cAMP assay, the media was carefully removed from the white-bottom plate and replaced with media containing 2% GloSensor cAMP reagent (Promega) and incubated at 37 °C for 2 hours. Cells were stimulated with various concentrations of α-MSH for 10 min and then the luminescence was read. Basal cAMP luminescence was subtracted, and cAMP values plotted as a percentage of maximum wildtype cAMP. Data is from 3 independent experiments (mean ± SEM).
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4

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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5

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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