Screen Quest™ Fluorimetric ELISA cAMP Assay Kit

1. Prepare samples
2. Add 75 µL/well of cAMP standard or test samples into the anti-cAMP coated 96-well plate
3. Incubate at room temperature for 5-10 mins
4. Add 25 µL/well of 1X HRP-cAMP Conjugate
5. Incubate at room temperature for 2 hours
6. Wash 4 times with 200 µL/well Washing Buffer
7. Add 100 µL/well of Amplite™ Red
8. Incubate at room temperature for 15 to 60 minutes
9. Monitor fluorescence increase at Ex/Em = 540/590 nm

Do not freeze Anti-cAMP Ab Pre-coated 96-well plate (Component H), store it at 4°C. Allow all the kit components to warm to room temperature before using them. Some material might be stick to the vial cap during the shipment. Briefly centrifuge the vial to collect all the content.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

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

Add 1 mL of Assay Buffer (Component B) to the vial of cAMP Standard (Component A).
Note        The unused cAMP stock solution (100 µM) should be aliquoted and stored at -20 °C.

Add 55 µL (Cat. # 36373) or 550 µL (Cat. # 36374) of Assay Buffer (Component B) into the vial of HRP-cAMP Conjugate (Component C).
Note        The unused 50X HRP-cAMP conjugate stock solution should be divided into single use aliquots and stored them at -20 °C.

Add 1 mL of 10X Wash Solution (Component D) to 9 mL distilled water.

Add 50 µL (Cat. # 36373) or 500 µL (Cat. # 36374) of DMSO to the vial of Amplite™ Red (Component G).
Note        0.5 µL of 200X Amplite™ Red stock solution is enough for one assay point. The unused reconstituted stock solution should be aliquoted and stored at -20 °C

Make 1:50 dilution with Assay Buffer (Component B) to have 1X HRP-cAMP conjugate working solution before use. Store it on ice or 4 °C.
Note        25 µL of 1X HRP-cAMP conjugate working solution is enough for one assay point; prepare appropriately volume for single use only.

Add 50 µL of Amplite™ Red stock solution (200X) and 11.5 µL of 3% H₂O₂ (Component F) into 10 mL of Substrate Buffer (Component I).
Note        The Amplite™ Red working solution is not stable, use it promptly.

1. Treat cells as desired:The following is an example of Hela cells treated with Forskolin to induce cAMP in a 96-well plate format: Aspirate off cell growth medium, add 100 µL/well 100 µM Forskolin in Hanks and 20 mM Hepes buffer (HHBS), incubate in a 5% CO₂, 37°C incubator for 15 minutes. Aspirate off cell solution after the incubation, add 100 µL/well of Cell Lysis Buffer (Component E), and incubate at room temperature for another 10 minutes. This cell lysate can be assayed directly or after diluted in Assay Buffer (Component B).
   
   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.
2. Tissue Samples:It is important to rapidly freeze tissues after collection (e.g., using liquid nitrogen) due to quick metabolism of cyclic nucleotides in tissue. Weigh the frozen tissue and add 10 - 20 µL/mg of cell lysis buffer. Homogenize the sample on ice. Spin at top speed for 5 minutes and collect the supernatant. The supernatant may be assayed directly.
3. Urine, Plasma and Culture Medium Samples:Urine and plasma may be tested directly with 1:200 to 1:1000 dilutions in 1X Lysis Buffer. Culture medium can also be tested with 1:10 to 1:200 dilutions in Lysis Buffer.
   
   Note        RPMI medium may contain > 350 fmol/µL cAMP.

1. All the assay wells will be prepared in the following orders: A) cAMP standards, control, or tests samples; B) HRP-cAMP Conjugate.
2. Add 75 µL/well of the cAMP diluted standard solution and test samples into each well of the anti-cAMP Ab coated 96-well plate (Component H). We recommended duplicating the assays for each standard and testing sample. Incubate at room temperature for 5 to 10 minutes.
3. Add 25 µL/well of 1X HRP-cAMP Conjugate working solution. Incubate at room temperature for 2 hours by placing the plate on shaker.
4. Aspirate plate contents, and wash 4 times with 200 µL/well of 1X wash solution.
5. Add 100 µL/well of Amplite™ Red working solution into each well, and incubate at room temperature for 10 mins to 2 hours, protected from light.
6. Monitor the fluorescence increase at Ex/Em = 540/590 nm (cutoff 570 nm) by using a fluorescence plate reader (top read mode).