Calbryte™ 520 AM
Overview | ![]() ![]() |
See also: Cell Signaling, Cellular Processes, Intracellular Ions, Physiological Probes, Flow Cytometry Reagents, Membrane Potential and Channels, G-Protein-Coupled Receptors (GPCR)
Molecular weight 1090.90 | Dissociation constant (Kd, nM) 1200 | Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
The intracellular calcium flux assay is a widely used method in monitoring signal transduction pathways and high throughput screening of G protein"coupled receptors (GPCRs) and calcium channel targets. Followed by Fluo-3 being introduced in 1989, Fluo-4, Fluo-8 and Cal-520 were later developed with improved signal/background ratio, and became the widely used Ca2+ indicators for confocal microscopy, flow cytometry and high throughput screening applications. However, there are still a few severe problems with Fluo-4. For example, as for Fluo-3, in all most all the intracellular calcium assays with Fluo-4 AM, probenecid is required to prevent the cell-loaded Fluo-4 from leaking out of cells. The use of probenecid with Fluo-4-based calcium assays compromises the assay results since probenecid is well-documented to have a variety of complicated cellular effects. Calbryte™ 520, AM is a novel fluorescent and cell-permeable indicator for the measurement of intracellular calcium. Like other dye AM esters, Calbryte™ 520 AM is non-fluorescent and non-activatable. Once Calbryte™ 520 AM enters the cell, it is readily hydrolyzed by intracellular esterase where it becomes activated and responsive to calcium. The activated indicator is now a polar molecule that is incapable of freely diffusing through the cell membrane, essentially trapping it inside the cell. Upon binding calcium ions, Calbryte™ 520 produces a bright fluorescence signal with extremely high signal/background ratio. It has the identical excitation and emission wavelength as Fluo-4, thus the same Fluo-4 assay settings can be readily applied to Calbryte™ 520-based calcium assays. Its greatly improved signal/background ratio and intracellular retention properties make Calbryte™ 520 AM the most robust indicator for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists in live cells.
Platform
Flow cytometer
Excitation | 488 nm laser |
Emission | 530/30 nm filter |
Instrument specification(s) | FITC channel |
Fluorescence microscope
Excitation | FITC |
Emission | FITC |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 490 |
Emission | 525 |
Cutoff | 515 |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
Example protocol
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.
Calbryte™ 520 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calbryte™ 520 AM in anhydrous DMSO.PREPARATION OF WORKING SOLUTION
Calbryte™ 520 AM Working Solution
On the day of the experiment, either dissolve Calbryte™ 520 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Calbryte™ 520 AM at a final concentration of 4 to 5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.Note The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Calbryte™ 520 AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ probenecid products, including water-soluble, sodium salt, and stabilized solution, can be purchased from AAT Bioquest.
SAMPLE EXPERIMENTAL PROTOCOL
Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
- Prepare cells in growth medium overnight.
- On the next day, add 1X Calbryte™ 520 AM working solution into your cell plate.
Note If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading. - Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines. - Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
- Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Calbryte™ 520 AM to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 91.667 µL | 458.337 µL | 916.674 µL | 4.583 mL | 9.167 mL |
5 mM | 18.333 µL | 91.667 µL | 183.335 µL | 916.674 µL | 1.833 mL |
10 mM | 9.167 µL | 45.834 µL | 91.667 µL | 458.337 µL | 916.674 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Images
Figure 1. ATP response was measured in CHO-K1 cells using Calbryte™ 520 AM (Cat No. 20653) and Fluo-4, AM (Cat No. 20550). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of either 10 µg/mL Calbryte™ 520 AM in HH Buffer with probenecid or 10 µg/mL Fluo-4, AM in HH Buffer with probenecid was added to the wells and incubated for 45 minutes at 37°C. Both dye loading solutions were removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added to achieve the final indicated concentration of 10 µM. Images were acquired on a Keyence microscope in the FITC channel.
Figure 2. Carbachol dose-response was measured in CHO-M1 cells with Calbryte™ 520 AM and Fluo-4 AM. CHO-M1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 520 AM in HH Buffer or 10 µg/ml Fluo-4 in HH Buffer was added and incubated for 45 minutes at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. Carbachol (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
Citations
View all 158 citations: Citation Explorer
The influence of spontaneous and visual activity on the development of direction selectivity maps in mouse retina
Authors: Tiriac, Alexandre and Bistrong, Karina and Pitcher, Miah N and Tworig, Joshua M and Feller, Marla B
Journal: Cell reports (2022): 110225
Authors: Tiriac, Alexandre and Bistrong, Karina and Pitcher, Miah N and Tworig, Joshua M and Feller, Marla B
Journal: Cell reports (2022): 110225
Downregulated Calcium-Binding Protein S100A16 and HSP27 in Placenta-Derived Multipotent Cells Induce Functional Astrocyte Differentiation
Authors: Cheng, Yu-Che and Huang, Chi-Jung and Ku, Wei-Chi and Guo, Shu-Lin and Tien, Lu-Tai and Lee, Yih-Jing and Chien, Chih-Cheng
Journal: Stem Cell Reviews and Reports (2022): 1--14
Authors: Cheng, Yu-Che and Huang, Chi-Jung and Ku, Wei-Chi and Guo, Shu-Lin and Tien, Lu-Tai and Lee, Yih-Jing and Chien, Chih-Cheng
Journal: Stem Cell Reviews and Reports (2022): 1--14
iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2
Authors: Dulloo, Iqbal and Atakpa-Adaji, Peace and Yeh, Yi-Chun and Levet, Cl{\'e}mence and Muliyil, Sonia and Lu, Fangfang and Taylor, Colin W and Freeman, Matthew
Journal: Nature Communications (2022): 1--18
Authors: Dulloo, Iqbal and Atakpa-Adaji, Peace and Yeh, Yi-Chun and Levet, Cl{\'e}mence and Muliyil, Sonia and Lu, Fangfang and Taylor, Colin W and Freeman, Matthew
Journal: Nature Communications (2022): 1--18
CPVT-associated calmodulin variants N53I and A102V dysregulate Ca2+ signalling via different mechanisms
Authors: Prakash, Ohm and Held, Marie and McCormick, Liam F and Gupta, Nitika and Lian, Lu-Yun and Antonyuk, Svetlana and Haynes, Lee P and Thomas, N Lowri and Helassa, Nordine
Journal: Journal of Cell Science (2022): jcs258796
Authors: Prakash, Ohm and Held, Marie and McCormick, Liam F and Gupta, Nitika and Lian, Lu-Yun and Antonyuk, Svetlana and Haynes, Lee P and Thomas, N Lowri and Helassa, Nordine
Journal: Journal of Cell Science (2022): jcs258796
Engineered Extracellular Matrices with Integrated Wireless Microactuators to Study Mechanobiology
Authors: Uslu, Fazil E and Davidson, Christopher D and Mailand, Erik and Bouklas, Nikolaos and Baker, Brendon M and Sakar, Mahmut Selman
Journal: Advanced Materials (2021): 2102641
Authors: Uslu, Fazil E and Davidson, Christopher D and Mailand, Erik and Bouklas, Nikolaos and Baker, Brendon M and Sakar, Mahmut Selman
Journal: Advanced Materials (2021): 2102641
Pharmic Activation of PKG2 Alleviates Diabetes-Induced Osteoblast Dysfunction by Suppressing PLC$\beta$1-Ca2+-Mediated Endoplasmic Reticulum Stress
Authors: Jia, Tingting and Wang, Ya-nan and Feng, Yao and Wang, Chenchen and Zhang, Dongjiao and Xu, Xin
Journal: Oxidative Medicine and Cellular Longevity (2021)
Authors: Jia, Tingting and Wang, Ya-nan and Feng, Yao and Wang, Chenchen and Zhang, Dongjiao and Xu, Xin
Journal: Oxidative Medicine and Cellular Longevity (2021)
Mechanistic Investigation of Cell Cryopreservation Aided by Raman Spectroscopy: Challenges and Solutions for hiPSC Technologies
Authors: Li, Rui
Journal: (2021)
Authors: Li, Rui
Journal: (2021)
NMDA receptor inhibition increases, synchronizes, and stabilizes the collective pancreatic beta cell activity: Insights through multilayer network analysis
Authors: {\v{S}}terk, Marko and Kri{\v{z}}an{\v{c}}i{\'c} Bombek, Lidija and Skelin Klemen, Ma{\v{s}}a and Slak Rupnik, Marjan and Marhl, Marko and Sto{\v{z}}er, Andra{\v{z}} and Gosak, Marko
Journal: PLoS Computational Biology (2021): e1009002
Authors: {\v{S}}terk, Marko and Kri{\v{z}}an{\v{c}}i{\'c} Bombek, Lidija and Skelin Klemen, Ma{\v{s}}a and Slak Rupnik, Marjan and Marhl, Marko and Sto{\v{z}}er, Andra{\v{z}} and Gosak, Marko
Journal: PLoS Computational Biology (2021): e1009002
Activation of the cytosolic calcium-independent phospholipase A2 $\beta$ isoform contributes to TRPC6 externalization via release of arachidonic acid
Authors: Putta, Priya and Smith, Andrew H and Chaudhuri, Pinaki and Guardia-Wolff, Rocio and Rosenbaum, Michael A and Graham, Linda M
Journal: Journal of Biological Chemistry (2021): 101180
Authors: Putta, Priya and Smith, Andrew H and Chaudhuri, Pinaki and Guardia-Wolff, Rocio and Rosenbaum, Michael A and Graham, Linda M
Journal: Journal of Biological Chemistry (2021): 101180
Influence of Acetylcholine Esterase Inhibitors and Memantine, Clinically Approved for Alzheimer's Dementia Treatment, on Intestinal Properties of the Mouse
Authors: Nguyen, Vu Thu Thuy and Sallbach, Jason and dos Santos Guilherme, Malena and Endres, Kristina
Journal: International journal of molecular sciences (2021): 1015
Authors: Nguyen, Vu Thu Thuy and Sallbach, Jason and dos Santos Guilherme, Malena and Endres, Kristina
Journal: International journal of molecular sciences (2021): 1015
References
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Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
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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
Journal: The International Journal of Biochemistry & Cell Biology (2017)
Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
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Journal: Neural Regeneration Research (2017): 945
Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
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Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
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Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
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