A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets

by Qin Zhao, Xing Han, Zhenjun Diwu, Jinfang Liao

Introduction

The Calcium influx assay is a preferred method for monitoring the activities of GPCR and calcium channels. We have developed a novel no wash probeniceid-free Cal 520™ Calcium Assay for cell-based calcium mobilization high-throughput screening assays. Cal 520™ AM is a new fluorescent calcium-sensitive dye developed for monitoring GPCR and calcium channel targets with a sifnigicantly improved signal to noise ration and intracellular retention.

Cal 520™ AM ester is non-fluorescent. Once it enters the cells, the lipophilic AM blocking groups are cleaved by intracellular esterases, resulting in a negatively charged fluorescent dye that stays inside cells.When cells are stimulated with bioactive compounds, the receptor signals release intracellular calcium. As the dye binds to Ca²⁺ inside the cells, fluorescence intensity is greatly enhanced.

Cal 520™ Calcium Assay Principle. [Ca²⁺] Increase via Gq or calcium channel is measured by Cal 520™ fluorescence enhancement.

In this study, the signal intensity and S/N (signal to background) ratio of probeniceid-free Cal 520™ Calcium Assay Kit, BD™ PBX Calcium Assay Kit, Fluo-4 Direct, and Calcium-4, along with Fluo-3 AM and Fluo-4 AM were evaluated with different receptor signaling pathways using several cell lines including CHO-M1, HEK-293 and Jurkat cells. Fluo-4 Direct, Calcium-4, Fluo-3 AM and Fluo-4 AM which are easily pumped out by organic-anion transporters. Probeniceid-free Cal 520™ Calcium Assay kit and Cal 520™AM has much better cellular retention than other existing calcium indicators in addition to its significantly higher S/N ration. It requires no organic-anion transporter inhibitors (such as probenecid) present in the assay system. Cal 520™ AM is an improved high S/N ration and better intracellular retention make the Cal 520™ calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.

Materials and Methods

CHO-M1 or HEK cells were plated at 96-well black wall/clear bottom costar plate at 37^oC incubator for overnight.
For Wash Assay: Take growth medium off, incubate the cells with Fluo-3 AM, Fluo-4 AM or Cal 520™ AM at 37^oC for 90 min, then at room temperature for 30 min.
For No Wash Assay: The cells were incubated with Fluo-4 Direct, Calcium 4, BD™ PBX Calcium Assay Kit, Cal 520 ™ NW kit or Cal 520™ PBC-free kit at 37^oC for 90 min, then at room temperature for 30 min.
Run calcium efflux experiments on Flexstation.

Method

Results

Response of endogenous P2Y receptor to ATP in CHO-M1 cells in the absence of probenecid. CHO-M1 cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Fluo-3 AM, Fluo-4 AM or Cal 520™ AM in HHBS were added into the wells, and the cells were incubated at 37°C for 2 hour. The dye loading medium were replaced with 100 µl HHBS, 50 µl of 300 µM ATP were added, and then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

ATP dose response on endogenous P2Y receptor in CHO-M1 cells with or without probenecid. CHO-M1 cells were seeded overnight in 50,000 cells per 100 µl per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed and every one third of the plate were incubated with 100 µl of 5 µM or Cal 520™ AM or Fluo-4 AM in HHBS with or without 2.5 mM probenecid, and the cells were incubated at 37°C for 2 hour. ATP (50 µL/well) was added using Flexstation to achieve the final indicated concentrations.

Carbachol dose response on enxogenous M1 receptor in CHO-M1 cells with or without probenecid. CHO-M1 cells were seeded overnight in 50,000 cells per 100 µl per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed and every one third of the plate were incubated with 100 µl of 5 µM or Cal 520™ AM or Fluo-4 AM in HHBS with or without 1 or 2.5 mM probenecid, and the cells were incubated at 37°C for 2 hour. ATP (50 µL/well) was added using Flexstation to achieve the final indicated concentrations.

Comparison of homogeneous calcium assays on response of endogenous P2Y receptor to ATP in CHO-M1 cells in the absence of probenecid. CHO-M1 cells were seeded overnight in 50,000 cells per 100 µl per well in a 96-well black wall/clear bottom costar plate. The cells were incubated with 100 µl of Cal 520™ PBC-free Ca Kit, Cal 520™ NW kit, BD™ PBX Ca kit, Fluo-4 direct or Calcium 4 in HHBS with or without 1 or 2.5 mM probenecid for 2 hours at 37^oC, 5% CO₂ incubator. ATP (50 µL/well) was added using Flexstation to achieve the final indicated concentrations.

Comparison of homogeneous calcium assays on response of endogenous P2Y receptor to ATP in HEK-293 cells. HEK-293 cells were seeded overnight in 30,000 cells per 100 µl per well in a 96 well black wall/clear bottom costar plate. The cells were incubated with 100 µl of 5 different calcium assay kits in HHBS for 2 hours at 37^oC, 5% CO₂ incubator. ATP (50 µL/well) was added using Flexstation to achieve the final indicated concentrations. The EC₅₀ of ATP for Cal 520 ™ PBC-free Ca Kit is 2 µM, Cal 520 ™ NW kit is 1 µM, BD™ PBX Ca kit is 8 µM, Fluo-4 direct is 5 µM, and Calcium 4 is 7 µM respectively.

Comparisons of homogeneous calcium assays on concanavalin A induced capacitative clacium entry (CCE) in JurKat cells. Jurkat cells were suspended at 2X10⁶ cells per ml in calcium-free HHBS buffer, cells were incubated with 100 µl of 5 different calcium assay kits in calcium-free HHBS buffer at 2X10⁵ cells/well/100 µL in a 96-well black wall/clear bottom poly-d lysine plate for 1.5 hr at 37^oC incubator. At the end of the 10 min incubation, 10 µL /well of channel opener concanavalin A at 3 µg/ml (final in well concentration) with or without channel inhibitor capsacin were added into the cells to achieve the final indicated concentrations. 50 µL/well of HBSS with additional 24 mM (5X) calcium was added using FlexStation.

Summary

Cal 520™ AM is a novel calcium indicator for measuring intracellular calcium. The high S/N ratio and good intracellular retention make the probenecid-free Cal 520™ calcium assay a robust tool for evaluating GPCR and calcium channel targets (especially for those interfered with probenecid), as well as for screening their agonists and antagonists. Cal 520™ Calcium Assay has the following benefits and features:

Enable probenecid-free Ca²⁺ assays.
Significantly higher S/N ratio than any other commercially available fluorescent Ca²⁺ assays.
Essentially identical spectra to Fluo-3™, Fluo-4™, and Fluo-8™.

Original created on February 10, 2012, last updated on February 10, 2012
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