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Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit
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.
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
Product family
| Name | Excitation (nm) | Emission (nm) |
| Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit | 581 | 593 |
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
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
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Journal: Pharmacological Research (2022): 106322
Selective activation of TRPA1 ion channels by nitrobenzene skin sensitizers DNFB and DNCB
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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)
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
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Authors: Endo, Ken-ichi and Sakamoto, Asuka and Fujisawa, Koushi
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References
View all 172 references: Citation Explorer
Calcium imaging of cortical neurons using Fura-2 AM
Authors: Barreto-Chang OL, Dolmetsch RE.
Journal: J Vis Exp. (2009)
Authors: Barreto-Chang OL, Dolmetsch RE.
Journal: J Vis Exp. (2009)
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
Authors: Bailey S, Macardle PJ.
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
Authors: Hirst RA, Harrison C, Hirota K, Lambert DG.
Journal: Methods Mol Biol (2006): 37
Load of calcium probe Fura -2/AM in Escherichia coli cells
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Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica
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