Screen Quest™ TR-FRET No Wash cAMP Assay Kit
Example protocol
CELL PREPARATION
Plate cells overnight in growth medium at 30,000 -100,000 cells/well for a 96-well plate.
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.
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 °C 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.
PREPARATION OF STOCK SOLUTIONS
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 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 °C.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/36379
PREPARATION OF WORKING SOLUTION
Add 50 µL of solution (Component A) to 2.5 mL of Cell Lysis Buffer (Component D).
Note: Make soultion just before use and as per needed.
Add 50 µL of solution (Component B) to 2.5 mL of Cell Lysis Buffer (Component D).
Note: Make soultion just before use and as per needed.
SAMPLE EXPERIMENTAL PROTOCOL
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.
BL | BL | TS | TS |
CS1 | CS1 | ... | ... |
CS2 | CS2 | ... | ... |
CS3 | CS3 | ||
CS4 | CS4 | ||
CS5 | CS5 | ||
CS6 | CS6 | ||
CS7 | CS7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
CS1-CS7 | 25 µL | Serial Dilution |
BL | 25 µL | Diluent (Component E) |
TS | 25 µL | Test Sample |
Table 3. Overview of the protocol
cAMP Standard | Cells | ||||
Negative Control | Positive Control | Standard Curve | Negative Control | Non-stimulated | Stimulated |
25 µL Diluent | 25 µL Diluent | 25 µL Standard | 25 µL cells | 25 µL cells | 25 µL cells |
25 µL Compound Buffer | 25 µL Compound Buffer | 25 µL Compound Buffer | 25 µL Compound Buffer | 25 µL Compound Buffer | 25 µL Compound |
Incubate 30 min at RT | |||||
25 µL Lysis Buffer | 25 µL cAMP-trFluor™ 650 working solution | 25 µL cAMP-trFluor™ 650 working solution | 25 µL Lysis Buffer | 25 µL cAMP-trFluor™ 650 working solution | 25 µL cAMP-trFluor™ 650 working solution |
25 µL Anti cAMP-trFluor™ Eu working solution | |||||
Incubate 30min at RT |
Table 4. Compatible HTRF® plate readers
Manufacturers | Instruments |
Berthhold Technologies | Tristar2 S; Mithras LB 940; Mithras2 LB 943 |
Hidex | Sense; Sense Beta Plus |
Molecular Devices | Spectra Max i3X; Spectramax Paradigm; Spectramax M5e; Spectramax 3 |
Thermo Scientific | Varioskan Lux |
Biotek | Synergy Neo2; Cytation 5; Cytation 3; Synergy H1; Synergy 2 |
BMG Labtech | PHERAstar; CLARIOstar; POLARstar Omega; Fluostar Omega |
Tecan | Spark 10M; Infinite M100 Pro; Infinite F500; Infinite F200 Pro |
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.
- 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.
- Incubate the reaction at room temperature for 30 minutes.
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.
- 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.
- Incubate the reaction at room temperature for 30 minutes.
- Read on a compatible TR-FRET reader.
Citations
Authors: Avanzato, D and Genova, T and Pla, A Fiorio and Bernardini, M and Bianco, S and Bussolati, B and Mancardi, D and Giraudo, E and Maione, F and Cassoni, P and others, undefined
Journal: Scientific Reports (2016)
Authors: Tomankova, Hana and Valuskova, Paulina and Varejkova, Eva and Rotkova, Jana and Benes, Jan and Myslivecek, Jaromir
Journal: Stress (2015)
References
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Journal: Handb Exp Pharmacol (2016): 29
Authors: Miazzi F, Hansson BS, Wicher D.
Journal: J Exp Biol (2016): 1798
Authors: Wang Z, Liu D, Varin A, Nicolas V, Courilleau D, Mateo P, Caubere C, Rouet P, Gomez AM, V and ecasteele G, Fischmeister R, Brenner C.
Journal: Cell Death Dis (2016): e2198