cAMP designer

Example of Bridge-It^® cAMP designer Assay for Attached Cells

The Bridge-It^® cAMP designer assay allows for the stimulation and measurement of cAMP in adherent monolayer cells attached to the wells of a tissue culture microplate. In the following example, HEK 293 cells were trypsinized and plated into the wells of a 96-well polystyrene tissue culture microplate at a density of 25,000 cells per well in 50 µl of culture media. The cells were allowed to attach to the bottom of the wells overnight. The media was removed the next day and the wells were washed with a buffered saline solution. The wash buffer was then removed and replaced with 50 µl of KRB-IBMX buffer. For stimulation, 1 µl of forskolin at different concentrations was added to each well and incubated and rotated gently for 15 minutes at ~25ºC. To quantify cAMP in each well, the forskolin buffer solution was removed, 100 µl of the designer Assay Solution was added to each well, and the microplate was incubated for 30 minutes at ~25ºC with rotation. The well contents were then transferred into a black 96-well microplate and the fluorescent signal was read using the settings for fluoresceine (~480 nm excitation, ~520 nm emission).

Cell Preparation and Forskolin Stimulation - HEK-293 cells were grown using standard cell culture conditions to ~70-80% confluency. The cells were trypsinized, harvested, and washed in serum-free Krebs Ringers Bicarbonate buffer containing a phosphodiesterase inhibitor (i.e., KRB-IBMX buffer). Cyclic AMP (cAMP) levels were measured following forskolin stimulation of the cells using the Bridge-It^® designer assay. Following incubation with Bridge-It^® Assay Solution for 30 min at ~ 25°C, the fluorescent signal was read at ~480 nm excitation, ~520 nm emission. Data analysis was performed using relative fluorescence (RF) as a signal change relative to a blank (RF= (F0-F)/F0). RF analysis has been shown to be highly reproducible and does not depend greatly on the instrument used to read fluorescence. Examples can be seen below.