Bridge-It®
cAMP Assays – Selectivity, Sensitivity
and Performance Characteristics
 The
Bridge-It® cAMP assay is highly specific.
ATP, AMP, and cGMP have all been tested for
selectivity using the cAMP assay. No response
was detected using the Bridge-It® cAMP
assay with any of these compounds within the
concentration range that would be expected
to occur in “real life” samples
(i.e., millimolar range for AMP and ATP, and
micromolar range for cGMP). The sensitivity
and performance characteristics of the Bridge-It®
cAMP designer
and the Bridge-It® all
in one assay products
are presented in the following Table and Figures.
Bridge-It®
cAMP |
Black
Microplate (recommended) |
cAMP
Detection Level |
designer
|
96-well |
5nM
(0.5 pmol/well in 100 µl vol.) |
all
in one |
384-well |
5nM
(100 fmol/well in 20 µl vol.) |
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.
Example of Bridge-It® cAMP designer
Assay for Cells in Suspension
|
