Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit

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4.0e+33.0e+32.0e+3100010.11e-21e-3- FLIPR Calcium 4- Fluo-4- Calbryte 520Data legend Generated with Quest Graph™ ATP (uM) RFU Hover mouse to interact
Comparison of fluorescent signal response of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. Calcium flux response was measured with Screen Quest™ Calbryte™ 520 Probenecid-Free and Wash-Free Calcium Assay Kit, FLIPR Calcium 4 Assay Kit , and Fluo-4 Direct Calcium Assay kit. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
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Unit Size: Cat No: Price (USD): Qty:
36317 $195


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

Ex/Em (nm)490/525
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader, FDSS, NOVOStar, FlexStation, ViewLux, IN Cell Analyzer, ArrayScan
Category GPCR
Calcium GPCR Assays
Related Calcium Channels
pH and Ion Indicators
Calcium flux assays are the preferred methods in drug discovery for screening G protein coupled receptors (GPCR). Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit provides the most robust homogeneous fluorescence-based assay for detecting the intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Calbryte™-520NW which can cross cell membrane. Calbryte™-520 NW is the brightest calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Calbryte™-520NW are cleaved by non-specific cell esterase, resulting in a negatively charged fluorescent dye that stays inside cells, and its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with screening compounds, the receptor signals release of intracellular calcium, which greatly increase the fluorescence of Calbryte™-520NW. The characteristics of its excellent cell retention, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium) make Calbryte™-520NW an ideal indicator for measurement of cellular calcium. Calbryte™-520NW is the only calcium dye that does not require probenecid for better cellular retention. This Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit provides the most optimized assay method for monitoring G-protein-coupled receptors (GPCRs) and calcium channels with fragile or difficult cell lines. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells in growth medium
  2. Add Calbryte™ 520 NW dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at room temperature or 37°C for 15-30 minutes
  4. Monitor fluorescence at Ex/Em = 490/525 nm

Important notes
Thaw all the kit components at room temperature before use.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:490 nm
Emission:525 nm
Cutoff:515 nm
Recommended plate:Solid black
Other Instruments: FDSS, NOVOStar, FlexStation, ViewLux, IN Cell Analyzer, ArrayScan
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Calbryte™ 520 NW stock solution:
Add 20 µL (Cat. # 36317) or 200 µL (Cat. # 36318 and # 36319) of DMSO into the vial of Calbryte™ 520 NW (Component A) and mix them well. Note: 20 µL of Calbryte™ 520 NW stock solution is enough for one plate. Unused Calbryte™ 520 NW stock solution can be aliquoted and stored at < -20 oC for more than one month if the tubes are sealed tightly. Note: Protect from light and avoid repeated freeze-thaw cycles.

2. Assay buffer (1X):
Mix 9 mL of HHBS (Component C, not included in the kit Cat. # 36319) with 1 mL of 10X Pluronic® F127 Plus (10X) (Component B) and mix them well.

Preparation of working solution

Calbryte™ 520 NW dye-loading solution:
Add 20 µL of Calbryte™ 520 NW stock solution into 10 mL of Assay Buffer (1X) and mix them well. Note: This working solution is stable for at least 2 hours at room temperature. Note: 10 mL dye-loading solution is enough for one 96-wells plate.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol
  1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Calbryte™ 520 NW dye-loading solution into the cell plate.

  2. Incubate the dye-loading plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 15 - 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation. If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1 hour (It is recommended that the incubation time be no longer than 2 hours.)

  3. Prepare the compound plate with HHBS or your desired buffer.

  4. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate ATP samples. We recommend using the Online Four Parameter Logistics Calculator which can be found at:

https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator

Figure 1. Comparison of fluorescent signal response of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. Calcium flux response was measured with Screen Quest™ Calbryte™ 520 Probenecid-Free and Wash-Free Calcium Assay Kit, FLIPR Calcium 4 Assay Kit , and Fluo-4 Direct Calcium Assay kit. ATP (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Yanjiao Wu, Xiaoli Xu, Lunkun Ma, Qian Yi, Weichao Sun, Liling Tang
Journal: The International Journal of Biochemistry & Cell Biology (2017)

Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
Authors: Jia Liu, Qing Du, He Zhu, Yu Li, Maodong Liu, Shoushui Yu, Shilei Wang
Journal: Int J Clin Exp Med (2017): 6861--6868

Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
Authors: Jiang Lu, Xue-qin Yao, Xin Luo, Yu Wang, Sookja Kim Chung, He-xin Tang, Chi Wai Cheung, Xian-yu Wang, Chen Meng, Qing Li
Journal: Neural Regeneration Research (2017): 945

Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
Authors: Gang Yang, Zhenghua Xiao, Xiaomei Ren, Haiyan Long, Kunlong Ma, Hong Qian, Yingqiang Guo
Journal: Scientific Reports (2017): 41781

Di (2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection
Authors: Xia Sun, Yi Lin, Qiansheng Huang, Junpeng Shi, Ling Qiu, Mei Kang, Yajie Chen, Chao Fang, Ting Ye, Sijun Dong
Journal: Journal of cellular and molecular medicine (2015): 581--594

The effect of mitochondrial calcium uniporter on mitochondrial fission in hippocampus cells ischemia/reperfusion injury
Authors: Lantao Zhao, Shuhong Li, Shilei Wang, Ning Yu, Jia Liu
Journal: Biochemical and biophysical research communications (2015): 537--542

Fungus induces the release of IL-8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways.
Authors: Xu-Dong Peng, Gui-Qiu Zhao, Jing Lin, Nan Jiang, Qiang Xu, Cheng-Cheng Zhu, Jain-Qiu Qu, Lin Cong, Hui Li
Journal: International journal of ophthalmology (2014): 441--447

Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells
Authors: Qing Li, Jiang Lu, Xianyu Wang
Journal: Neural regeneration research (2014): 2002

Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats
Authors: Nan Liang, Peng Wang, Shilei Wang, Shuhong Li, Yu Li, Jinying Wang, Min Wang
Journal: Journal of Neural Transmission (2014): 593--600

Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve--mast cell interaction in a hapten-induced mouse model of atopic dermatitis
Authors: M Hagiyama, T Inoue, T Furuno, T Iino, S Itami, M Nakanishi, H Asada, Y Hosokawa, A Ito
Journal: British Journal of Dermatology (2013): 771--778