Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

cAMP standard (1mM):
Add 100 µL Diluent (Component E) to cAMP Standard (Component C) and mix them well. Note: The unused cAMP standard can be aliquoted and stored at -20^oC.

For convenience, use our Serial Dilution Planner

1. Anti cAMP-trFluor™ Eu working solution:
Add 55 µL (Cat. # 36379) or 550 µL (Cat. # 36380 or # 36381) of Diluent (Component E) into the vial of Anti cAMP-trFluor™ Eu (Component A). Add 50 µL of reconstituted solution to 2.5 mL of Cell Lysis Buffer (Component D). Note: Make soultion just before use and as per needed.

2. cAMP-trFluor™ 650 working solution:
Add 55 µL (Cat. # 36379) or 550 µL (Cat. # 36380 or # 36381) of Diluent (Component E) into the vial of cAMP-trFluor™ 650 (Component B). Add 50 µL of reconstituted solution to 2.5 mL of Cell Lysis Buffer (Component D). Note: Make soultion just before use and as per needed.

Cell Preparation:

For adherent cells: Plate cells overnight in growth medium at 30,000 -100,000 cells/well for a 96-well plate.

For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellets in culture medium at 100,000-300,000 cells/well for a 96-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiment.

Treat cells as desired: The following is an example for Hela cells treated with Forskolin to induce cAMP in a 96-well plate format. 25µL cells in growth medium, add 25 µL/well 100 µM Forskolin in Hanks and 20 mM Hepes buffer (HHBS), incubate in a 5% CO₂, 37 ^oC incubator for 15 minutes. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. Cells may be seeded the day before or on the day of the experiment depending upon the cell type and/or the effect of the test compounds.

Table 1. Layout of cAMP standards and test samples in a solid black 96-well microplate. CS = cAMP standard (CS1-CS7); BL = blank control; TS = test sample.

Table 2. Reagent composition for each well.

cAMP assay in cell lysate

1. Prepare and add cAMP standards (CS), blank controls (BL) and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 12.5 µL of each corresponding reagent instead of 25 µL. Note: Test samples could be Non-stimulated and/or stimulated samples.
2. Add 25 µL of treatment (Compound resuspended in buffer) into each well of cAMP standard, blank control, and test samples to make the total cAMP assay volume of 50 µL/well. For a 384-well plate, add 12.5 µL of working solution into each well for a total volume of 25 µL/well.
3. Incubate the reaction at room temperature for 30 minutes.
4. Add 25 µL of cAMP-trFluor™ 650 working solution into each well of cAMP standard, blank control, and test samples to make the total cAMP assay volume of 75 µL/well. For a 384-well plate, add 12.5 µL of working solution into each well for a total volume of 37.5 µL/well. Note: For negative controls, Lysis Buffer can be added.
5. Add 25 µL of cAMP-trFluor™ Eu working solution into each well of cAMP standard, blank control, and test samples to make the total cAMP assay volume of 100 µL/well. For a 384-well plate, add 12.5 µL of working solution into each well for a total volume of 50 µL.
6. Incubate the reaction at room temperature for 30 minutes.
7. Read on a compatible TR-FRET reader.

Overview of the protocol:

Table 3. Compatible HTRF® plate readers

Results are Relative Fluorescence Units at 665nm and 620nm. Ratio is calculated as the F_665nm / F_620nm ratio and expressed in Δ F%.

R= F_665nm/F_620nm

Δ  F%= 100% x (R _sample-R _neg)/R_neg

Draw a standard curve by plotting Δ F% versus cAMP concentration.