Applications - Application Notes

A Cost-effective Workflow for High-Throughput Screening of GProtein Coupled Receptors (GPCRs) - Monitoring Receptor Mediated Calcium Flux

03-Jun-11

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Related Products: EL406

 

 

Authors: Paul Held and Peter Banks, BioTek Instruments, Inc., Winooski, VT; Dee Shen, JoAnne Schultz, and Wayne Patton, Enzo Life Sciences, Farmingdale, NY

 

Enzo Life Sciences

 

Drugs targeting members of the GPCR super-family represent the core of modern medicine, accounting for the majority of the best-selling drugs and roughly 40% of all prescription pharmaceuticals on the market today. We describe a new fluorescent probe for drug discovery which is provided to cells as a cell membrane-permeable ester. Intracellular calcium binding to the fluorescent probe is readily measured using a fluorescence microplate reader equipped with a dual-reagent dispenser. This fluorescence-based assay workflow is suitable for monitoring calcium mobilization across a broad spectrum of biological targets. The easy-to-use protocol is automation-friendly and can be performed in a convenient 96- well or 384-well microplate format. The described assay workflow results in low sampleto- sample variability and excellent Z' values in a miniaturized format.

 

Introduction

 

Cell-based assays that monitor the functional activation of GPCRs are considered a critical part of the drug discovery process. Many of these receptors initiate signaling cascades that can result in changes in calcium levels within the cell [1]. GPCRs are a super-family of transmembrane receptor genes the exact number of which is unknown but has been suggested to consist of over 1000 proteins [2]. GPCRs are integral membrane proteins characterized by an extracellular N-terminus, followed with seven transmembrane a-helices connected by three intracellular and three extracellular loops and an intracellular C-terminus [2]. The GPCR system is modular, consisting of an a-subunit which binds and hydrolyzes GTP, as well as b and γ subunits which form a complex. The complex is modular and with each subunit having several different subtypes, the mix and match of which provides much of the functional versatility of the GPCRs. The a-subunits can be divided into four families of which the Gaq/ Ga11 family interacts with phospholipase C, leading to the mobilization of intracellular calcium [2].

The calcium ion is an important second messenger involved in many physiological and signaling processes within cells. Fluo-3 & Fluo-4 dyes are widely used calcium ion indicators for in-cell measurement of agonist-stimulated and antagonist-inhibited calcium signaling in high-throughput screening applications.

However their relatively weak fluorescent signals have limited their application in some challenging cell lines and with certain membrane receptors. We report upon a new calcium-sensitive fluorescent dye, referred to as FluoForte® reagent, which provides superior performance relative to the conventional dyes employed in such assays.

 

 Schematic diagram depicting FluoForte internalization and calcium mobilization resulting from GPCR activation

Figure 1. Schematic diagram depicting FluoForte internalization and calcium mobilization resulting from GPCR activation

 

FluoForte® dye is added to cells in the form of a non-fluorescent AM ester. Once inside cells, the lipophillic AM blocking groups are cleaved by nonspecific cellular esterases, resulting in negatively charged fluorescent dye that is retained within the cells. The fluorescence intensity of the dye is greatly enhanced upon binding to calcium.

When cells are stimulated with active screening compounds, the GPCR-mediated signaling cascade releases stores of intracellular calcium, which lead to greatly increased fluorescence signals (Figure 1).

The Synergy™ Mx instrument (BioTek Instruments) is a multifunctional microplate reader with liquid handling and kinetic fluorescent reading capability. This study validates the instrument as a low-cost platform for performing cellbased, fast-kinetic assays, such as intracellular calcium mobilization measurements with FluoForte® reagent. The instrument provides a unique dispensing system that facilitates addition of small amounts of agonist or antagonist (<10 μL) to cells, followed by instantaneous reading.

 

 FluoForte dye Assay Process.

Figure 2. FluoForte dye Assay Process.

 

Materials and Methods

 

Hela, CHO-K1 and CHO-M1 cell lines used in these studies were obtained from ATCC. FluoForte® calcium assay kits were provided by Enzo Life Sciences (Farmingdale, NY). Assays were performed according to the assay kit instructions. Cells were seeded overnight at 40,000 cells per well (100 μL) in Corning 3603 black sided clear bottom plates. The following day the medium was removed and the cells were incubated with 100 μL of 4 μM Fluo- Forte® AM dye in HBSS in a 5% CO2 cell culture incubator for 1 hour. The plates were placed in the Synergy Mx Microplate Reader (BioTek Instruments) and ATP (20 μL/ well) was added using the reader’s syringe pump dispenser to achieve desired final concentrations. Wells were read using a 485 excitation and 528 emission wavelengths. Readings were taken kinetically for 90 seconds and the maximal fluorescence values plotted.

 

Results

 

Hela cells that have been incubated with FluoForte® AM dye exhibit a large increase in fluorescence intensity as observed by fluorescence microscopy when treated with 1 μM ATP (Figure 2). Prior to the addition of ATP all of the cells show a low degree of fluorescence, indicating that the FluoForte® AM ester has been internalized and converted to the active form by cellular esterases.

 

Increase in fluorescence intensity with addition of ATP.

 

Figure 3. Increase in fluorescence intensity with addition of ATP. Images of HeLa cell (A) with and (B) without treatment with 1 μM ATP. Cells were treated with ATP, washed twice with HBSS then immediately imaged with a fluorescence microscope (Carl Zeiss Inc) using an FITC filter set.

 

Optimal cell loading time for FluoForte® dye was examined using a time course study of intracellular calcium mobilization in CHO-K1 cells. Incubating with Fluo- Forte® dye for 60 minutes at 37°C in a 5% CO2 incubator provided a maximal signal (Figure 4). The signal gain difference observed between loading times of 30 and 45 minutes is most likely the result of increasing amounts of dye being converted by cellular esterases into an actively fluorescent product. Data from a 45 minute loading time suggested that little was to be gained by increasing the loading time beyond 60 minutes.

 

Time course study of FluoForte® dye detection of intracellular calcium mobilization in CHO-K1 cells

 

Figure 4. Time course study of FluoForte® dye detection of intracellular calcium mobilization in CHO-K1 cells. CHO cells were seeded overnight in 40,000 cells per 100 μL per well in a 96-well black wall/clear bottom plate. The cells were incubated with 100 μL of 4 μM FluoForte® reagent for 15-45 minutes at room temperature or 60 min at 37°C. ATP (20 μL/well) was added using a BioTek Synergy™ Mx reader syringe pump dispenser to achieve a final concentration of 400 nM.

 

The FluoForte® calcium assay kit was automated using an EL406™ Combination Washer Dispenser (BioTek Instruments). Carbachol stimulation of the M3-muscarinic receptor expressed in CHO-M1 cells demonstrates a dose dependent increase in fluorescence when measured with FluoForte® reagent (Figure 5).

Automated Assay with carbachol dose response curves in CHO-M1 cells, expressing M3-muscarinic receptor and graph statistics.

Figure 5. Automated Assay with carbachol dose response curves in CHO-M1 cells, expressing M3-muscarinic receptor and graph statistics.

 

Using this automated system the assay displays a large assay window with a high (>0.5) Z’ factor (Figure 5). In addition the EC50 value agrees with data previously determined running the assay manually (Data not shown).

Comparison of manual and automated assay with ATP dose response curves in CHO-M1 cells, expressing P2Y endogenous receptors and graph statistics.

Figure 6. Comparison of manual and automated assay with ATP dose response curves in CHO-M1 cells, expressing P2Y endogenous receptors and graph statistics. Wash steps and reagent addition were carried out using an EL406 Combination Washer Dispenser to automate the liquid handling tasks.

 

When a manual protocol and the automated protocol are directly compared, very similar results are obtained. CHO-M1 cells expressing endogenous P2Y receptors were stimulated with increasing concentrations of ATP. When normalized fluorescence signals were plotted, identical curves are observed (Figure 6). Both protocols have a large assay window with a greater than 2.5 fold increase in signal intensity upon maximal stimulation (Table 2). The high Z’ values calculated for both methods and very similar EC50 results suggest that either method can be used with a high degree of confidence.

FluoForte® dye is significantly brighter than Fluo4 dye. When kinetic measurements of CHO-K1 cells stimulated with 400 nM ATP are plotted FluoForte® dye exhibits a slightly higher initial degree of fluorescence than Fluo4, as a result of basal levels of intracellular calcium (Figure 7). More importantly, with calcium mobilization FluoForte® dye signal increases to a greater degree than Fluo4 and the signal is sustained longer. This is corroborated by larger signal to blank ratios (S/B) with ATP and histamine stimulation of CHO-K1 and Hela cells respectively (Figures 8 and 9).

Comparison of FluoForte® & Fluo-4 dye-based detection of intracellular calcium mobilization in CHO-K1 cells.

Figure 7. Comparison of FluoForte® & Fluo-4 dye-based detection of intracellular calcium mobilization in CHO-K1 cells. Cells were seeded as previously described and then incubated with 4 μM of either FluoForte® or Fluo-4 dye and then stimulated with 400 nM ATP. Response after the addition of ATP was measured kinetically using a Synergy™ Mx reader (BioTek Instruments).

The increased brightness of FluoForte® dye as compared to Fluo4 has significant experimental advantages. The brighter signal results in greater fluorescence at the EC50 and maximal response concentrations (Figure 8). This results in superior Z’ values at the EC50 concentration and an increase in the S/B ratio when CHO-K1 cells expressing endogenous P2Y receptors are stimulated with ATP (Figure 8).

FluoForte® and Fluo-4 dye ATP dose response curves in CHO-K1 cells, expressing P2Y endogenous receptors with curve statistics.

Figure 8. FluoForte® and Fluo-4 dye ATP dose response curves in CHO-K1 cells, expressing P2Y endogenous receptors with curve statistics. Cells were seeded as previously described and then incubated with 4 μM of either FluoForte® or Fluo-4 dye for 1 hr at 37°C and then stimulated with ATP at the indicated concentrations.

Histamine dose response curves in Hela cells, expressing Histamine H1 receptors and graph statistics.

Figure 9. Histamine dose response curves in Hela cells, expressing Histamine H1 receptors and graph statistics. Cells were seeded as previously described and then incubated with 4 μM of either FluoForte® or Fluo-4 dye for 1 hr at 37°C and then stimulated with histamine at the indicated concentrations.

Similar results are observed with Hela cells expressing the histamine H1 receptor with histamine stimulation. With both fluorescence dyes equivalent EC50 are observed, suggesting that the degree of calcium mobilization is equivalent (Figure 9). However, the increased signal response of FluoForte® dye results in greater S/B ratio at maximal response and improved Z’ factor values at the EC50.

Discussion

These data demonstrate the utility of the FluoForte® calcium indicator dye for monitoring changes in calcium flux as a result of GPCR stimulation. Numerous GPCRs result in a rapid increase in intracellular calcium concentration, which can be measured using FluoForte® dye. Fluo- Forte® dye is a fluorogenic ester that easily penetrates the cell membrane. Once inside the cell, ubiquitous cellular esterases hydrolyze the compound, which generates a fluorescent anionic compound that is trapped within the cell. Because only the intracellular compound is fluorescent, any change in fluorescence can be attributed to intracellular changes in calcium ion concentration.

It should be noted that some cell lines express organic anion transporters or the MDR phenotype, which leads to the export of negatively charged Fluo-Forte® dye. This same phenomenon occurs with Fluo3 and Fluo4 dyes. The MDR phenotype is the up-regulation of a family of transmembrane ATP binding cassette (ABC) transporter proteins that are present in practically all living organisms [3-5]. These proteins cause chemotherapy resistance in cancer by actively extruding a wide variety of therapeutic compounds from the malignant cells. This phenomenon can easily be prevented by adding Dye Efflux Inhibitor, an anion transporter inhibitor to the assay wells.

BioTek Instruments provides an ideal instrument platform on which to run this assay. The BioTek Synergy™ Mx Microplate Reader provides a cost effective system to perform fast kinetic, cell-based assays, such as the Fluo- Forte® calcium mobilization assay. The system’s unique pipetting system allows for instantaneous reading upon agonist stimulation. When the FluoForte® reagent is employed on the BioTek platform, analysis of both G protein- coupled receptor and calcium ion channel targets is readily accomplished. The easy-to-use protocol does not require a wash step or the addition of a quencher dye, which could potentially modify pharmacological parameters. FluoForte® dye can be loaded at 37°C or room temperature, which makes it amenable to high throughput screening applications in drug discovery. As demonstrated, removal of culture medium can be performed using the EL406™ Combination Microplate Washer Dispenser without compromising assay quality, but significantly simplifying assay workflow.

The Synergy Mx instrument, in combination with the Fluo- Forte® Calcium Assay Kit, offers researchers an integrated instrument-reagent combination that provides high-performance results at an affordable price.

 

References

  1. Filmore, D. (2004) It’s a GPCR World, Modern Drug Discovery Nov, 2004:24-28
  2. Wettschureck, N. and S. Offermanns (2005) Mammalian G Proteins and Their Cell Type Specific Functions, Physiological Reviews 85:1159-1204.
  3. Gupta RS. (1988) Intrinsic multidrug resistance phenotype of Chinese hamster (rodent) cells in comparison to human cells. Biochem. Biophys. Res. Commun. 153:598- 605.
  4. Hunter J., Hirst BH., Simmons NL. (1991) Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein. Br. J. Cancer 64:437-444.
  5. Bodey B., Taylor CR., Siegal SE., and Kaiser HE. (1995) Immunocytochemical observation of multidrug resistance (MDR) p170 glycoprotein expression in human osteosarcoma cells. The clinical significance of MDR protein overexpression. Anticancer Res. 15(6B):2461-2468.

 

 

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