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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.

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.

Cal 520™ Calcium Assay Principle

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

 

Materials and Methods


  1. CHO-M1 or HEK cells were plated at 96-well black wall/clear bottom costar plate at 37oC incubator for overnight.
  2. For Wash Assay: Take growth medium off, incubate the cells with Fluo-3 AM, Fluo-4 AM or Cal 520™ AM at 37oC for 90 min, then at room temperature for 30 min.
  3. 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 37oC for 90 min, then at room temperature for 30 min.
  4. Run calcium efflux experiments on Flexstation.

 

Results


Figure1.  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.

 

Figure 2.  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.

 

Figure 3.  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.

 

Figure 4.  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 37oC, 5% CO2 incubator. ATP (50 µL/well) was added using Flexstation to achieve the final indicated concentrations.

Figure 5.  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 37oC, 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.

Figure 6.  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 37oC 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
Tagged under: Calcium GPCR Analysis, Calcium Imaging, Calcium Mobilization