Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit
Ordering information
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Related products
Overview | SDSProtocol |
See also: Calcium Indicators, Cell/Cytoplasmic Membrane Potential Activity & Analysis, Cell Signaling Pathways, Intracellular Ions, Screen Quest™ Products
Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
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™-520 AM which can cross cell membrane. Calbryte™-520 AM is the brightest calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Calbryte™-520 AM 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™-520 AM. The characteristics of its excellent cell retention, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium) make Calbryte™-520 AM an ideal indicator for measurement of cellular calcium. Calbryte™-520 AM 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.
Platform
Fluorescence microplate reader
Excitation | 490 nm |
Emission | 525 nm |
Cutoff | 515 nm |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
Other instruments
ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLuxComponents
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells in growth medium
- Add Calbryte™ 520 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at room temperature or 37 °C for 30-60 minutes
- Monitor fluorescence at Ex/Em = 490/525 nm
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
PREPARATION OF STOCK SOLUTIONS
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.
Note 20 µL of Calbryte™ 520 AM stock solution is enough for one plate. Unused Calbryte™ 520 AM stock solution can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly.
Note Protect from light and avoid repeated freeze-thaw cycles.
1. Calbryte™ 520 AM stock solution
Add 20 µL (Cat. # 36317) or 200 µL (Cat. # 36318 and # 36319) of DMSO into the vial of Calbryte™ 520 AM (Component A) and mix them well.Note 20 µL of Calbryte™ 520 AM stock solution is enough for one plate. Unused Calbryte™ 520 AM stock solution can be aliquoted and stored at < -20 °C 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 AM dye-loading solution
Add 20 µL of Calbryte™ 520 AM 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.
SAMPLE EXPERIMENTAL PROTOCOL
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Calbryte™ 520 AM dye-loading solution into the cell plate.
- Incubate the dye-loading plate in a cell incubator for 30-60 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.)
Note Do NOT wash the cells after dye loading. - Prepare the compound plate with HHBS or your desired buffer.
- Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 490/525 nm.
Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Product Family
Name | Excitation (nm) | Emission (nm) |
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit | 581 | 593 |
Images
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.
Citations
View all 16 citations: Citation Explorer
OPEN ACCESS EDITED BY
Authors: Taggart, Clifford and Kesturu, Girish S and Brown, Ryan and Wu, Xiaojun and Wang, Zhengtao and Chen, Z and Wang, G and Xie, X and Liu, H and Liao, J and others,
Journal: Oral neutrophils-the good, the bad, and the ugly (2023): 41
Authors: Taggart, Clifford and Kesturu, Girish S and Brown, Ryan and Wu, Xiaojun and Wang, Zhengtao and Chen, Z and Wang, G and Xie, X and Liu, H and Liao, J and others,
Journal: Oral neutrophils-the good, the bad, and the ugly (2023): 41
Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB
Authors: Wu, Han and Niu, Canyang and Qu, Yaxuan and Sun, Xiaoying and Wang, KeWei
Journal: Journal of Biological Chemistry (2022)
Authors: Wu, Han and Niu, Canyang and Qu, Yaxuan and Sun, Xiaoying and Wang, KeWei
Journal: Journal of Biological Chemistry (2022)
Preferential Gs protein coupling of the galanin Gal1 receptor in the $\mu$-opioid-Gal1 receptor heterotetramer
Authors: De Oliveira, Paulo A and Moreno, Estefan{\'\i}a and Casajuana-Martin, Nil and Casad{\'o}-Anguera, Ver{\`o}nica and Cai, Ning-Sheng and Camacho-Hernandez, Gisela Andrea and Zhu, Hu and Bonifazi, Alessandro and Hall, Matthew D and Weinshenker, David and others,
Journal: Pharmacological Research (2022): 106322
Authors: De Oliveira, Paulo A and Moreno, Estefan{\'\i}a and Casajuana-Martin, Nil and Casad{\'o}-Anguera, Ver{\`o}nica and Cai, Ning-Sheng and Camacho-Hernandez, Gisela Andrea and Zhu, Hu and Bonifazi, Alessandro and Hall, Matthew D and Weinshenker, David and others,
Journal: Pharmacological Research (2022): 106322
Selective activation of TRPA1 ion channel by skin sensitizer nitrobenzene DNFB
Authors: Wu, Han and Qu, Yaxuan and Sun, Xiaoying and Wang, Kewei
Journal: Authorea Preprints (2021)
Authors: Wu, Han and Qu, Yaxuan and Sun, Xiaoying and Wang, Kewei
Journal: Authorea Preprints (2021)
Diquafosol tetrasodium elicits total cholesterol release from rabbit meibomian gland cells via P2Y 2 purinergic receptor signalling
Authors: Endo, Ken-ichi and Sakamoto, Asuka and Fujisawa, Koushi
Journal: Scientific reports (2021): 1--8
Authors: Endo, Ken-ichi and Sakamoto, Asuka and Fujisawa, Koushi
Journal: Scientific reports (2021): 1--8
Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels
Authors: Zhou, Qiqi and Shi, Yuntao and Qi, Hang and Liu, Huijie and Wei, Ningning and Jiang, Yong and Wang, KeWei
Journal: The FASEB Journal (2020): 12338--12353
Authors: Zhou, Qiqi and Shi, Yuntao and Qi, Hang and Liu, Huijie and Wei, Ningning and Jiang, Yong and Wang, KeWei
Journal: The FASEB Journal (2020): 12338--12353
Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry & Cell Biology (2017)
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
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Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
Journal: Neural Regeneration Research (2017): 945
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
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: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781
Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781
Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
Authors: Liu, Jia and Du, Qing and Zhu, He and Li, Yu and Liu, Maodong and Yu, Shoushui and Wang, Shilei
Journal: Int J Clin Exp Med (2017): 6861--6868
Authors: Liu, Jia and Du, Qing and Zhu, He and Li, Yu and Liu, Maodong and Yu, Shoushui and Wang, Shilei
Journal: Int J Clin Exp Med (2017): 6861--6868
References
View all 172 references: Citation Explorer
Calcium imaging of cortical neurons using Fura-2 AM
Authors: Barreto-Chang OL, Dolmetsch RE.
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Authors: Barreto-Chang OL, Dolmetsch RE.
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A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
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Journal: J Immunol Methods (2006): 220
Measurement of [Ca2+]i in whole cell suspensions using fura-2
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Journal: Methods Mol Biol (2006): 37
Load of calcium probe Fura -2/AM in Escherichia coli cells
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Journal: Wei Sheng Wu Xue Bao (2005): 805
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Journal: Wei Sheng Wu Xue Bao (2005): 805
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Authors: McConnell G, Riis E.
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Authors: McConnell G, Riis E.
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Comparison of human recombinant adenosine A2B receptor function assessed by Fluo-3-AM fluorometry and microphysiometry
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Application notes
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Introducing Calbryte™ Series
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
Evaluation of FLIPR Calcium Assays for Screening GPCR and Calcium Channel Targets
Fluorescent Dye AM Esters
Introducing Calbryte™ Series
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
Evaluation of FLIPR Calcium Assays for Screening GPCR and Calcium Channel Targets
Fluorescent Dye AM Esters
FAQ
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?
How do I make an AM ester stock solution?
How do you measure Ca2+ response with calcium indicators like Calbryte 520, potassium salt?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
How do I make an AM ester stock solution?
How do you measure Ca2+ response with calcium indicators like Calbryte 520, potassium salt?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?