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Calbryte™ 520 AM

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.
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.
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.
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Price ()
Catalog Number20650
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)1200
Molecular weight1090.90
SolventDMSO
Spectral properties
Excitation (nm)493
Emission (nm)515
Quantum yield0.751
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Related products
Calbryte™-520L AM
Calbryte™-520L, potassium salt

OverviewpdfSDSpdfProtocol


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

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC
EmissionFITC
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateBlack 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.
  1. Prepare cells in growth medium overnight.
  2. 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.
  3. 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.
  4. 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.
  5. 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 mg0.5 mg1 mg5 mg10 mg
1 mM91.667 µL458.337 µL916.674 µL4.583 mL9.167 mL
5 mM18.333 µL91.667 µL183.335 µL916.674 µL1.833 mL
10 mM9.167 µL45.834 µL91.667 µL458.337 µL916.674 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)493
Emission (nm)515
Quantum yield0.751

Images


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 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.
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.
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
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
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
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
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
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)
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
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
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

References


View all 63 references: Citation Explorer
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)
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
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
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
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: Sun, Xia and Lin, Yi and Huang, Qiansheng and Shi, Junpeng and Qiu, Ling and Kang, Mei and Chen, Yajie and Fang, Chao and Ye, Ting and Dong, Sijun
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: Zhao, Lantao and Li, Shuhong and Wang, Shilei and Yu, Ning and Liu, Jia
Journal: Biochemical and biophysical research communications (2015): 537--542
Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats
Authors: Liang, Nan and Wang, Peng and Wang, Shilei and Li, Shuhong and Li, Yu and Wang, Jinying and Wang, Min
Journal: Journal of Neural Transmission (2014): 593--600
Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells
Authors: Li, Qing and Lu, Jiang and Wang, Xianyu and others, undefined
Journal: Neural regeneration research (2014): 2002
Fungus induces the release of IL-8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways.
Authors: Peng, Xu-Dong and Zhao, Gui-Qiu and Lin, Jing and Jiang, Nan and Xu, Qiang and Zhu, Cheng-Cheng and Qu, Jain-Qiu and Cong, Lin and Li, Hui
Journal: International journal of ophthalmology (2014): 441--447
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: Hagiyama, M and Inoue, T and Furuno, T and Iino, T and Itami, S and Nakanishi, M and Asada, H and Hosokawa, Y and Ito, A
Journal: British Journal of Dermatology (2013): 771--778

Documents


Certificate of origin
Safety data sheet
Product protocol
Certificate of analysis

Application notes


A New Robust Fluorescent Calcium Indicator for Ca2+ Flux Assays in Living Cells
Calbryte™ 520, Calbryte™ 590 and Calbryte™ 630 Calcium Detection Reagents
Introducing Calbryte™ Series
The Eight Best Green Fluorescent Calcium Indicators
What's A Ratiometric Indicator

FAQ


Are there any calcium indicators that don't require probenecid (PBC)?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
What is a calcium flux assay?
What is a calcium ion?
What is an AM ester?
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