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.) |
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 either the Bridge-It®
all in one
assay or 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 all
in one Assay for Cells in Suspension
The
Bridge-It® cAMP all
in one 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 all
in one 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).
Example
of Bridge-It® cAMP all
in one Assay for Attached Cells
|
