Complete cAMP workflow solution using the CatchPoint cAMP Fluorescent Assay Kit

Introduction

In this study, we demonstrate how the CatchPoint^®cAMP Fluorescent Assay Kitcombined with the SpectraMax^®i3 MultiMode Detection Platform can be used to monitor the response of HEK293 cells to forskolin, an activator of adenylate cyclase (Figure 1).

Figure 1: CatchPoint cAMP workflow

G-Protein Coupled Receptors (GPCRs) are important transmembrane proteins that translate extracellular signals into intracellular responses. These intracellular responses are comprised of cell-signaling cascades that initiate changes in protein activity and expression within the cell [1].

Cyclic adenosine 3’, 5’-monophosphate (cAMP) is a secondary messenger that works downstream of GPCRactivation. Upon ligand binding to a GPCR, a conformational change occurs, activating the receptor and in turn activating a G protein. Further signal transduction depends on the type of G protein activated. Activation of G_sleads to upregulation of cAMP by adenylate cyclase, which activates protein kinase A, causing phosphorylation of targets involved in processes such as dopamine signaling, gluconeogenesis, vasodilation, as well as mitogenesis and oocyte maturation [2–6].

The CatchPoint cAMP FluorescentAssay Kit measures cAMP levels via a competitive immunoassay (Figure 2). The assay requires only a single washing step, and readings can be taken in as little as 10 minutes or as long as 24 hours following substrate addition.

Figure 2: CatchPoint cAMP assaymechanism. Unlabeled cAMP produced by cells competes with cAMP-HRP conjugate for binding to anti-cAMP antibodies. Increasing amounts of cellular cAMP are detected through decreasing HRP activity.

Materials

• CatchPoint cAMP Fluorescent Assay Kit(Molecular Devices cat. #R8088)
• HEK293 cells (ATCC cat. #CRL-1573)
• Krebs-Ringer bicarbonate buffer - KRGB buffer (Sigma cat. #K4002)
  • Sodium bicarbonate (Sigma, cat. #S5761)
• Phosphate-buffered saline (PBS, Life Technologies cat. #10010)
• Phosphodiesterase inhibitor, 3-isobutyl-1- methylxanthine (IBMX, Sigma cat. #I7018)
• 3% hydrogen peroxide (H₂0₂) solution
• Minimum Essential Medium with Earle’s salts and L-glutamine (Corning cat. #10-010)
  • Fetal bovine serum (Gemini Bio-Products cat. #100-106)
  • Penicillin-streptomycin (Life Technologies cat. #15070-063)
  • Forskolin (Sigma cat. #F6886)
  • Poly-D-Lysine Coated 96-well Microplates (Corning cat. #354413)
  • SpectraMax i3 Multi-Mode Microplate Reader
  • SpectraMax^®MiniMax^™300 Imaging Cytometer
  • MultiWash+^™Microplate Washer

Methods

HEK293 cells were cultured in complete growth medium (MEM + 10% FBS + 1% Pen/ Strep) and grown to 80-90% confluence in T75 flasks. Cells were harvested using 0.05% trypsin, and 25,000 cells were seeded in each well of a poly-D-lysine coated, 96-well, black-wall, clear-bottom microplate (25,000-100,000 cells/well is an acceptable range). Cells were allowed to adhere for at least 18 hours in a 37°C incubator with 5% CO₂.

On the day of assay, cell confluence was measured using the transmitted light channel of the SpectraMax MiniMax 300 Imaging Cytometerand StainFree^™Cell Detection Technology, which enables accurate measurement of cell counts or percent area covered by cells (confluence) for quality control purposes (Figure 3). Rows of cells with high cell confluence and low inter-well variability were selected for the CatchPoint assay. Cells were pre-stimulated with 0.75 mM IBMX for 10 minutes at room temperature. They were then treated with a dilution series of forskolin starting at 1,000 µM with a 1:3 dilution series for 15 minutes at 37°C. Cells were then lysed according to the CatchPoint assay protocol.

Figure 3: HEK293 cell percent confluence determined using the MiniMax cytometer and StainFree analysis. Rows A and C had similar cell confluence and the lowest variability between wells and were selected for assay.

The CatchPoint assay was performed as described in the product insert. A cAMP calibrator curve was performed to verify assay performance, as well as provide a means of calculating the amount of cAMP in cellular assay samples. Washing was performed on the MultiWash+ Microplate Washer. Samples were read on a SpectraMax i3 Multi-Mode Microplate Reader 30 minutes after StopLight Red substrate addition. All data analysis and curve fitting were performed with SoftMax^®Pro Software. A preconfigured protocol is available in the software’s protocol library.

Results

To assess cell confluence before stimulation, the MiniMax cytometer and StainFree technology were used to measure percent cell coverage in wells of the microplate (Figure 3). Rows A and C were chosen for the assay due to consistent percent confluence from well to well. Triplicate samples were taken from each treated well and assayed.

Figures 4 and 5 show results for the CatchPoint cAMP calibrator curve and cell-based assay. Both curves were generated using the 4-parameter curve fit in SoftMax Pro Software. The calibrator curve had an EC₅₀of 3.3 nM, which agreed closely with previous published data [7]. The Z’ factor was 0.91.

Figure 4: cAMP calibrator curve. EC₅₀was 3.3 nM, similar to previously published results. Samples were run in duplicate.

Figure 5: Cell-based assay performed with HEK293 cells. A forskolin concentration-response curve was run, starting at 1,000 µM with a 3-fold dilution series, with samples run in duplicate. EC₅₀was 2.3 µM.

The forskolin concentration-response curve had an EC₅₀of 2.3 µM, which is consistent with expected results for HEK293 cells assayed in an adherent format

Conclusion

The CatchPoint cAMP FluorescentAssay Kit allows for accurate measurements of GPCRactivity through cAMP detection. High signal stability (10 minutes to 24 hours), excellent Z’ factor, and the ability to be performed on plate readers with a fluorescence intensity detection mode make the CatchPoint assay a versatile choice for high-throughput screening.

Compatible with these Molecular Devices systems

References

1. Zaccolo, Manuela. “cAMP signal transduction in the heart: understanding spatial control for the development of novel therapeutic strategies.” British Journal of Pharmacology 158.1 (2009): 50-60.
2. Hanoune, Jacques, and Nicole Defer. “Regulation and role of adenylyl cyclase isoforms.” Annual Review of Pharmacology and Toxicology 41.1 (2001): 145-174.
3. Griendling, Kathy K., et al. “Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.” Arteriosclerosis, Thrombosis, and Vascular Biology 20.10 (2000): 2175-2183.
4. Kemp, Bruce E., et al. “Dealing with energy demand: the AMP-activated protein kinase.” Trends in Biochemical Sciences 24.1 (1999): 22-25.
5. Etgen, Anne M., Michael A. Ansonoff, and Arnulfo Quesada. “Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology.” Hormones and Behavior 40.2 (2001): 169-177.
6. Chini, Eduardo N., et al. “Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway.” Biochemical and Biophysical Research Communications 215.3 (1995): 868-873.
7. Hesley, Jayne, Janet Daijo, and Anne T. Ferguson. “Stable, sensitive, fluorescence-based method for detecting cAMP.” BioTechniques 33.3 (2002): 692-694.

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