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Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation

Simon Lydford & Petra Averbeck, Molecular Devices Limited, U.K.


Introduction
The FlexStation scanning fluorometer integrates sensitive optics, fluid transfer and temperature control, making it ideal for kinetic, cell-based fluorometric assays, such as the measurement of intracellular calcium, membrane potential and intracellular pH.

Calcium (Ca2+) is the most common signal transduction element in cells ranging from bacteria to specialized neurons. Measurement of changes in the concentration of intracellular cations, for example Ca2+, is important in understanding the mechanisms of many cellular processes1. A range of events such as receptor-ligand interactions mediate the elevation of intracellular calcium ([Ca2+]i) levels from ~100nM to ~1mM. This can trigger a number of events, including the release of synaptic transmitters, muscle contraction, hormone secretion, cell movements or apoptosis.

Changes in [Ca2+]i can be measured either with single-wavelength dyes (e.g. Fluo-3 and the FlexStation Calcium Assay Kit) or dual-wavelength ratiometric indicators, such as Fura-2. With single-wavelength dyes the fluorescence intensity is dependent on the Ca2+ concentration whilst ratiometric indicators undergo a spectral shift that is proportional to the Ca2+ concentration. The most common single-wavelength indicators, such as Fluo-3, are excited with visible light and the most common dual-wavelength dyes (Fura-2) require UV light2.

The FlexStation system incorporates a Xenon-lamp light source and dual monochromators that permit the use of essentially all dual-wavelength dyes for functional cellular assays. Although visible-light excitable Ca2+ indicators offer benefits such as reduced interference from sample auto fluorescence and higher absorbance by the dye (permitting the use of lower dye concentrations), ratiometric dyes offer several advantages over conventional single-wavelength indicators:

Benefits of using the 340/380 nm excitation ratios for Fura-2:
Signal is not dependent on:
-Dye concentration
-Illumination intensity
-Optical path length

The effects of uneven dye loading, dye leakage and photo bleaching are minimized
-Detection can usually be made over a period of 1 hour without significant loss of fluorescence

A more stable signal and better signal-to-noise ratio may be obtained
- Increased sensitivity

Accurate measurements of the intracellular Ca2+ concentration is possible

An alternative approach is a series of no-wash reagents available from Molecular Devices, such as the FlexStation Calcium Assay Kit. These, too, confer a number of potential advantages over conventional Ca2+ indicators.

Problems in a conventional assay protocol that may be reduced with no-wash reagents:
Cells removed from the plates during the washing procedure
Reduced responsiveness (competence) of the cells following washing
Variation in the residual volume of wash buffer, leading to variation in the concentration of test compound
The drop in fluorescence (resulting from the dilution of residual extracellular dye) upon addition of the test compound

In this application note we demonstrate the use of both single-wavelength and dual-wavelength Ca2+ indicators in the FlexStation benchtop scanning fluorometer and the possible benefits of a homogeneous assay system.


BACKGROUND
CHO-M1 cells contain endogenous nucleotide receptors (P2Y2)3,4 that are linked to activation of phospholipase C, generation of inositol phosphates (IPs), activation of protein kinase C (PKC) and release of intracellular Ca2+ stores5. Purinoceptors are expressed in the vast majority of cells and tissues, where they control a wide range of physiological processes. To date, five subtypes of the P2Y receptor6 have been identified and clonedP2Y1, P2Y2, P2Y4, P2Y6 and P2Y11. The principal physiological ligands for the P2Y2-receptor are UTP and ATP; they show similar potencies7. One important functional role of the P2Y2-receptor is the control of chloride ion fluxes in airway epithelia and mucociliary clearance in the lung, and thus may play a role in cystic fibrosis and other airway disorders.

Novel ligands targeting P2Y-receptors represent potential therapeutic agents and the ability to screen such compounds reliably and reproducibly has been demonstrated using FlexStation8.

Using Z-factor analysis9 and comparison of EC50 estimates and signal magnitude, we show the benefits of ratiometric Ca2+ dyes and homogeneous assay formats over conventional Ca2+ indicators.


METHODS
Cell Preparation
CHO cells (passage number > 40) were routinely grown in 75 cm2 flasks as a monolayer in Hams-F12 medium supplemented with 10% foetal bovine serum and 5 g/mL G418, and maintained at 37˚C in a CO2 incubator. Cells were passaged 1:10 every 2-3 days.

Measurement of [Ca2+]i
CHO cells were seeded into black-walled, clear-base 96-well plates (Costar UK) at a density of ~30,000 cells per well in Hams-F12 medium, supplemented as above and cultured overnight. The cells were then incubated at 37C for 60 minutes with the appropriate calcium indicator; Fluo-3, Fura-2 or FlexStation Calcium Assay Kit; and 2.5 mM probenecid (see appendix). The cells loaded with Fluo-3 or Fura2 were washed manually (BioHit 8-channel multipipette) three times with Hanks BSS medium containing 2.5 mM probenecid. Cells loaded with the FlexStation Calcium Assay Kit did not require this wash step. The final volume in each well was 200 mL.

The cell plates were then placed into the FlexStation system to monitor fluorescence before and after the addition of ATP (50 ml addition, ATP made at 5X final concentration, final concentrations 30 nm-30 mM). Experimental parameters are tabulated below.


Data Analysis
Responses were measured as peak fluorescence intensity minus basal fluorescence intensity (Fluo-3 or FlexStation calcium reagent) or the 340/380 nm excitation ratios, calculated within SoftMax Pro, for Fura-2. Data are expressed as mean s.e. mean and n > 12.

Individual sets of E/[A] curve data were fitted to a four-parameter logistic of the form:

where α, β, EC50 and m are the asymptote, bottom of curve, location (expressed as -log10 EC50 ) and slope parameters respectively. [A] is the concentration of the agonist where, in this case, its ATP. E is the relative fluorescence units or ratio of fluorescence units for ratiometric dyes. All curve-fitting routines were carried out using GraphPad Prism 3.0 (GraphPad Software, Inc.).

Z-factors were calculated using the formula:


where αc+ denotes standard deviation of the positive control and αc- represents standard deviation of the negative control. The term |c+ c- | denotes the absolute value of the difference between the mean of the positive control and the mean of the negative control. A Z-factor > 0.5 indicates a large separation band between the negative and positive controls and an excellent assay.


RESULTS

ATP elicited a concentration-dependent increase in [Ca2+]i in CHO cells (Figure 1) with similar EC50 values for cells loaded with Fluo-3, Fura-2 and FlexStation Calcium Assay Kit, respectively (Table 2).

One-way Analysis of Variance (ANOVA) indicated that the EC50 estimates were not significantly different from one another (P value = 0.62).


These results indicate that there is negligible difference in the ATP EC50 values from cells loaded with the three fluorescent reagents; however, there was a significant difference between the peak fluorescent intensities of the two single-wavelength indicators. With Fluo-3 there was an increase in signal of ~3,200 RFU above basal (1.8 fold increase) whilst with the FlexStation Calcium Assay Kit the increase in signal was 32,000 RFU (5.6 fold increase, see Table 1). Peak response tended to be more variable with Fluo-3 (see Figure 1). With Fura-2 there was a 4.8 fold increase in signal above basal.

The screening coefficient window (Z-factor), which reflects the dynamic range of the signal and the data variation for the assay9 was calculated using the buffer addition (negative control) and the 30 M ATP addition data (positive control). Z-factors obtained with Fura-2 and the FlexStation Calcium Assay Kit were 0.60 and 0.68 respectively (Table 1). This shows a large separation band between the negative and positive controls and a reproducible, high quality assay. The Zfactor obtained for the Fluo-3 data was 0.28, representing a useable assay. This probably reflects the less than optimal assay protocol and subsequent small dynamic range of the assay.


SUMMARY
In this study, ATP caused a concentration-dependent increase in [Ca2+]i in CHO cells endogenously expressing the P2Y2-receptor, with a potency similar to that reported previously. Furthermore, we have demonstrated that the FlexStation can be used to generate reliable, reproducible data for calcium mobilization assays used to monitor such Gq-coupled GPCRs.

The EC50 estimates for ATP were similar, whether the fluorescent dye Fluo-3 (0.639uM) or Fura-2 (0.639uM) or the FlexStation Calcium Assay Kit (0.651uM) was used, and were consistent between assay plates on the same day, as well as between assays analyzed on different days (less than 0.6 log unit differences). There were, however, significant differences in the dynamic ranges from the three different loading protocols, which is reflected in the corresponding Z-factors. Less than a twofold increase in signal above basal was observed when the cells were loaded with Fluo-3. The most likely cause is the subsequent wash steps in the protocol, reducing cell responsiveness. Using cells of a lower passage number and a dedicated cell-washer (Denley or similar) would probably have improved the Z-factor for Fluo-3 loaded cells. However, under these experimental conditions there was a 5-fold increase in signal observed with Fura-2, despite the cells undergoing a number of wash steps. This could be attributed to the beneficial ratiometric properties of the dye. However, the biggest fold increase in signal and Z-factor was observed with the FlexStation Calcium Assay Kit indicating that this "no-wash", homogeneous assay can lead to stronger signals, higher assay precision and, thus, improved physiological data.

In conclusion, FlexStation provides an easy to use, versatile tool for pharmacological and drug screening laboratories and has proven to be suitable for kinetic, cell-based fluorometric assays, such as the measurement of intracellular calcium. Additionally, FlexStation benefits from the ability to use ratiometric dyes to help improve data fidelity, although the Molecular Devices homogeneous assay kits have been shown to achieve the same goal with fewer steps in the assay.


ACKNOWLEDGEMENTS
Many thanks to Dr. Frank Hafner for his valuable opinion and consulting contribution in this study.


REFERENCES
1. Berridge, M.J. (1997). Elementary and global aspects of calcium signalling. J. Physiol., 499, 291 - 306.

2. Haughland, R.P. (2000). Molecular Probes Handbook of Fluorescent Probes and Research Chemicals. Chapter 20 - Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions.

3. Endogenous GPCR List by Agi Schonbrunn, Ph.D. Department of Integrative Biology, Pharmacology and Physiology, University of Texas Health Science Center, Houston, Texas. (http://www.tumor-gene.org/GPCR/gpcr.html)

4. Strassheim, D. and Williams, C.L. (2000). P2Y2 Purinergic and M3 Muscarinic Acetylcholine Receptors Activate Different Phospholipase C- Isoforms That Are Uniquely Susceptible to Protein Kinase C-dependent Phosphorylation and Inactivation. J. Biol. Chem., 275, 39767-39772.

5. Conant, A.R. et al (1998). Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304. Br. J. Pharmacol., 125, 357-364.

6. Communi, D., Robaye, B. & Boeynaems, J.M. (1999). Pharmacological characterization of the human P2Y11 receptor. Br. J. Pharmacol., 128, 1199-1206.

7. Law, C., Lydford, S.J. & Henderson, D.J. (1998). Characterisation of the P2receptor(s) on ECV304 endothelial cells using FLIPR. Naunyn-Schmeidebergs Arch.Pharmacol. 358, R126.

8. Zambon, A.C., Conklin, B.R., Brunton, L.L., Insel, P.A. & Ferguson, A.T. (2001). Application Note #1. Gq Protein Coupled Receptor Assays Using FlexStation. (http://www.moleculardevices.com/pdfs/flexstation_1.pdf).

9. Zhang, J-H., Thomas, T.D.Y. and Oldenburg, K. R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screening, 4, 6773.


APPENDIX
Materials
Cells : CHO cells (M1-WT3) - adherent Chinese Hamster Ovary cells stably expressing the rM1-muscarinic receptor (ECACC Ref. #: 940822109)

Reagents: FlexStation Calcium Assay Kit (Molecular Devices, cat # R8041), ATP (Adenosine 5'-triphosphate disodium salt, Sigma, cat # A2383), probenecid (Sigma, cat # P8761), DMSO, low water content (Sigma, cat # D2650), Fluo-3 AM ester (Molecular Probes, cat # F1242), Fura-2 AM ester (Molecular Probes, cat # F1221) and 20% pluronic acid solution (Molecular Probes, cat # P3000)

Plasticware : 75cm2 culture flasks (Corning cat # 430641), 96-well black walled, clear bottomed plates (Costar, cat # 3603), 96-well clear, U-bottom polypropylene plates (Greiner Bio-One, cat # 650101), Pipette tips for FlexStation - 200 L non-sterile, polypropylene tips (Molecular Devices, black cat # 9000-0622 or clear cat # 9000-0623)

Media and buffers : Hams-F12 medium (Invitrogen Life Technologies, cat # 31765-027), Hank's Balanced Salt Solution (HBSS) (10X) (Invitrogen Life Technologies, cat # 14065-049), HEPES 1M buffer solution (Invitrogen Life Technologies, cat # 15630-056), FBS: (Fetal Bovine Serum, heat inactivated, Invitrogen Life Technologies, cat # 10099-141), Geneticin (G418, Invitrogen Life Technologies, cat # 10131-019).


Fluo-3 and Fura-2 reagent preparation
Step 1 Wash buffer: this buffer contains a final concentration of 1X HBSS and 20 mM HEPES diluted in sterile d.H2O. Important: To prevent dye extrusion by the anion exchange protein, the wash buffer is supplemented with 2.5 mM probenecid.

Step 2 Prepare a fresh stock of 250 mM probenecid. Dissolve 710 mg probenecid in 5 mL of 1 M NaOH then add 5 mL of wash buffer and agitate vigorously.

Step 3 Fluo-3 and Fura-2 stock solutions. Add 25 L DMSO to 50 mg Fluo-3 AM or Fura-2 AM (final concentration ~2 mM).

Step 4 Dye loading buffer. Mix 25 L Fluo-3 or Fura-2 stock solutions and 25 L 20% pluronic acid solution, add this to 12 mL wash buffer containing 2.5 mM probenecid then add 120 L FBS. This prepares enough reagent for approximately one 96-well plate. Pluronic acid is used to enhance dye solubility and uptake.


Calcium assay kit reagent preparation
Step 1 Prepare 1X Reagent Buffer using component B and Loading Buffer by diluting component A with 1X Reagent Buffer as described in the instructions provided with the kit

Step 2 Prepare a fresh stock of 250 mM probenecid as above.

Step 3 Add 1:50 dilution of 250 mM probenecid to the Loading Buffer (final concentration 5 mM) and 1:100 dilution of 250 mM probenecid to 1X Reagent Buffer (final concentration 2.5 mM).


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