Cisbio cAMP HiRange (62AM6PEC) Assay: 384-well
Microplates
Regular height, 384-well, white, opaque, with lids
Reagent
1 mM IBMX-PBS: to resuspend cells and dilute compounds and forskolin
IBMX: 3-Isobutyl-1-methylxanthine, Sigma I5879
Cell preparation
Suspension cells: Lift cells with trypsin or non-enzymatic cell stripper, neutralize with growth medium and count cells. Spin down cells and resuspend in 1 mM IBMX-PBS at a desired concentration (see considerations).
Adherent cells: Seed cells at a desired density 24 hrs prior to the assay (see considerations).
Treatment (20 無 total)
Gs-coupled receptor assay: For suspension cells, add 15 無 cells to 384-well white plates + 5 無 4x agonist, incubate 20 min at 37C. For adherent cells, add 15 痞 of 1mM IBMX-PBS before compound addition.
Gi-coupled receptor assay: For suspension cells, add 10 無 cells to 384-well white plates + 5 無 4x agonist, incubate 5 min at room temperature, add 5 無 4x forskolin, incubate 20 min at 37蚓. For adherent cells, add 10 痞 of 1mM IBMX-PBS before compound additions.
Adjust reagent volumes when using antagonists.
Termination (40 無 final)
10 無 D2 (cAMP-d2, 1:20 in lysis buffer), followed by 10 無 K (Anti-cAMP-Cryptate, 1:20 in lysis buffer), incubate 60 min at room temperature, and read on FlexStation 3.
cAMP standards
Prepare during cell treatment, using 1 mM IBMX-PBS.
Add 20 μL/well to the plate before D2 and K additions.
| Standard |
Preparation |
Working (μM) |
Final (μM) |
Log[cAMP (M)] |
| Stock |
Reconstituted
reagent in water |
11.2 |
- |
- |
| A |
40 μL Stock + 40 μL Buffer |
5.6 |
2.8 |
-5.552842 |
| B |
16 μL A + 64 μL Buffer |
1.12 |
0.56 |
-6.251812 |
| C |
16 μL B + 64 μL Buffer |
0.224 |
0.112 |
-6.950782 |
| D |
16 μL C + 64 μL Buffer |
0.0448 |
0.0224 |
-7.649752 |
| E |
16 μL D + 64 μL Buffer |
0.00896 |
0.00448 |
-8.348722 |
| F |
16 μL E + 64 μL Buffer |
0.00179 |
0.0009 |
-9.047692 |
| G |
16 μL F + 64 μL Buffer |
0.00036 |
0.00018 |
-9.746662 |
| H |
64 μL Buffer |
0 |
0 |
-11 |
Considerations
Cell density optimization for suspension cells: Gs-coupled receptor: test 5-30 K/well; Gi-coupled receptor, test 2-6 K/well.
Cell density optimization for adherent cells: Gs-Coupled receptor: test 8-10K/well; Gi-Coupled receptor: test 6-8K/well
Forskolin concentration: Perform forskolin dose response experiment for each cell line, and test EC50-80 to achieve optimal inhibition by receptor agonist. Receptor-expressing cells could be very different from the parental cells. Forskolin EC50 may differ with treatment temperature and time.
Be cautious when changing microplate type and/or total reaction volume.
