logo
AAT Bioquest

Cal-520®, AM

ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
Response of endogenous P2Y receptor to ATP in CHO-K cells. CHO-K cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Cal 520 ™ AM in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 1 hour. The dye loading mediums were replaced with 100 µl HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using FITC channel before and after adding 50 µl of 300 µM ATP .
Two-photon calcium responses to tonal stimuli recorded at 140 ms intervals.<strong>&nbsp;</strong>Averaged traces (mean and S.E.M.) of ∆F/F0 in 44 neurons stained with Cal-520 AM. The red trace represents responses to 20 kHz stimuli lasting for 7s, and the blue trace shows responses to 20 kHz stimuli lasting for 1s in the same neurons. The off-responses to stimuli lasting for 7 s were significantly larger than the on-responses to stimuli lasting for 1 s (P&lt;0.0001). Source: <strong>Auditory cortical field coding long-lasting tonal offsets in mice</strong> by Baba et al., <em>Scientific Reports</em>, Sep. 2016.
Functional sperm analysis. (a) Tracks for freely swimming wildtype Prm2+/+ and heterozygous Prm2+/&minus; sperm. (b) Flagellar waveform. Sperm were tethered with their heads to a glass surface and the flagellar waveform was analyzed. One beat cycle was projected. Scale bar: 10&thinsp;&mu;m. (c) Changes in the intracellular Ca<sup>2+</sup> concentration in Prm2+/+, Prm2+/&minus;, and Prm2&minus;/&minus; sperm. Sperm have been loaded with Cal520-AM and stimulated with K8.6 (blue), 10&thinsp;mM 8-Br-cAMP (red), 10&thinsp;mM NH4Cl (green), or 2&thinsp;&mu;M ionomycin (light blue). Experiments have been measured using the stopped-flow technique. (d) Loading of sperm with Cal520-AM. Loading of Prm2+/&minus;, and Prm2&minus;/&minus; sperm was tested using fluorescence microscopy. Scale bar&thinsp;=&thinsp;20&thinsp;&mu;m. Source: <strong>Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile</strong> by Schneider et al.,&nbsp;<em>Scientific Reports</em>, Nov. 2016.
Selectivity of V1 neurons. A) Neurons stained with Cal-520 but not with SR-101 in the V1 of a wild-type mouse (left) and a Pcdh&alpha;1,12 mouse (right). The image was obtained using a two-photon microscope. B) Sample traces of neuronal calcium responses to moving grating patterns in eight directions (from -45&deg; to 270&deg; in 45&deg; steps) for 2 s in a wild-type mouse (left) and a Pcdh-&alpha;1,12 mouse (right). C) Cumulative distributions of the orientation selectivity index (OSI, left) and direction selectivity index (DSI, right) of neurons obtained from three wild-type mice and three Pcdh-&alpha;1,12 mice. The OSI was obtained from1698 and 1342 neurons, respectively. The DSI was obtained from 365 and 302 neurons with an OSI &gt; 0.45, respectively. There was no significant difference in the cumulative distribution of the OSI or DSI between wild-type and Pcdh&alpha;1,12 mice. Source: <strong>Molecular diversity of clustered protocadherin-&alpha; required for sensory integration and short-term memory in mice </strong>by Yamagishi et al., <em>Scientific Reports</em>, June 2018.
Ordering information
Price
Catalog Number
AvailabilityIn stock
Unit Size
Quantity
Add to cart
Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Physical properties
Dissociation constant (Kd, nM)320
Molecular weight1102.95
SolventDMSO
Spectral properties
Excitation (nm)492
Emission (nm)515
Quantum yield0.751
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
Related products
Cal-520®-Dextran Conjugate *MW 3,000*
Cal-520®-Dextran Conjugate *MW 10,000*
Cal-520®-Biotin Conjugate
Cal-520®-Biocytin Conjugate
Cal-520® NHS Ester
Cal-520®, sodium salt
Cal-520®, potassium salt
Cal-520FF™, potassium salt
Cal-520N™, potassium salt
Cal-520L®-Dextran Conjugate *MW 10,000*
Cal-520L™ maleimide
Chemical Phosphorylation Reagent I (CPR I)
Cell Meter™ Mitochondrial Hydroxyl Radical Detection Kit *Red Fluorescence*
Cal Green™ 1, hexapotassium salt
Cal Green™ 1, AM [Equivalent to Calcium Green-1, AM]
Cal-590™-Dextran Conjugate *MW 3,000*
Cal-590™-Dextran Conjugate *MW 10,000*
Cal-590™, sodium salt
Cal-590™, potassium salt
Cal-630™, sodium salt
Cal-630™, potassium salt
Cal-630™-Dextran Conjugate *MW 3,000*
Cal-630™-Dextran Conjugate *MW 10,000*
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*
Fluo-4, Pentapotassium Salt
Cal Red™ R525/650 potassium salt
Cal Red™ R525/650 AM
Fluo-3, AM *CAS 121714-22-5*
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*
Fluo-3, AM *Bulk package* *CAS 121714-22-5*
Fluo-3FF, AM *UltraPure grade* *Cell permeant*
Fluo-3, pentasodium salt
Fluo-3, pentapotassium salt
Fluo-3, pentaammonium salt
Fluo-3FF, pentapotassium salt
Fluo-8®, AM
Fluo-8®, sodium salt
Fluo-8®, potassium salt
Fluo-8H™, AM
Fluo-8H™, sodium salt
Fluo-8L™, AM
Fluo-8L™, sodium salt
Fluo-8L™, potassium salt
Fluo-8FF™, potassium salt
Fluo-8FF™, AM
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit
Mag-Fluo-4 potassium salt
Mag-Fluo-4 AM
Fluo-2, potassium salt
Fluo-2, AM
Fluo-5F, AM *Cell permeant*
Fluo-5F, pentapotassium Salt *Cell impermeant*
Fluo-5N, AM *Cell permeant*
Fluo-5N, pentapotassium Salt *Cell impermeant*
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit
Calbryte™ 520, potassium salt
Calbryte™ 590 AM
Calbryte™ 590, potassium salt
Calbryte™ 630 AM
Calbryte™ 630, potassium salt
Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit
Screen Quest™ Calbryte-590 Probenecid-Free and Wash-Free Calcium Assay Kit
Calbryte™-520L AM
Calbryte™-520L, potassium salt
Cal-500™, potassium salt
Cal-670™, potassium salt
Cal-670™-Dextran Conjugate *MW 3,000*
Cal-670™-Dextran Conjugate *MW 10,000*
Cal-770™, potassium salt
Cal-770™-Dextran Conjugate *MW 3,000*
Cal-770™-Dextran Conjugate *MW 10,000*
Calbryte™-520XL azide
Calbryte™-520XL, potassium salt
Calbryte™-520XL AM
Calbryte™-520XL-Dextran
RatioWorks™ Cal-520L®/Cy5-Dextran Conjugate *MW 10,000*
RatioWorks™ Cal-520®/zFluor 647™ -Dextran Conjugate *MW 10,000*
Cal-590L® Dextran Conjugate *MW 10,000*
RatioWorks™ Cal-590L®/Cy5-Dextran Conjugate *MW 10,000*
Show More (70)

OverviewpdfSDSpdfProtocol


Molecular weight
1102.95
Dissociation constant (Kd, nM)
320
Excitation (nm)
492
Emission (nm)
515
Quantum yield
0.751
Cal-520® AM provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. Cal-520® AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520® AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal-520™ AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520®. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520® AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520® calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.

Platform


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

Cal-520® AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Cal-520® AM in high-quality, anhydrous DMSO.

    Note: When reconstituted in DMSO, Cal-520® AM is a clear, colorless solution.

PREPARATION OF WORKING SOLUTION

Cal-520® AM Working Solution
  1. On the day of the experiment, either dissolve Cal-520® AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Cal-520® AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Cal-520® AM at a final concentration of 4-5 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 Cal-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 solutions, 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 Cal-520® AM working solution to 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 1 to 2 hours.

    Note: Incubating the dye for longer than 2 hours 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 Cal-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 mM90.666 µL453.33 µL906.659 µL4.533 mL9.067 mL
5 mM18.133 µL90.666 µL181.332 µL906.659 µL1.813 mL
10 mM9.067 µL45.333 µL90.666 µL453.33 µL906.659 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)492
Emission (nm)515
Quantum yield0.751

Product Family


NameExcitation (nm)Emission (nm)Quantum yield
Cal-520® maleimide4925150.751
Cal-520FF™, AM4925150.751
Cal-520N™, AM4925150.751
Cal-520® amine4925150.751
Cal-520® azide4925150.751
Cal-520® alkyne4925150.751
Cal-590™ AM5745880.621
Cal-630™ AM6096260.371
Calbryte™ 520 AM4935150.751
Cal-500™ AM3884820.481
Mag-520™ AM506525-
SoNa™ 520 AM491511-
Show More (3)

Images


Citations


View all 521 citations: Citation Explorer
Osteo-inductive effect of piezoelectric stimulation from the poly (l-lactic acid) scaffolds
Authors: Das, Ritopa and Le, Duong and Kan, Ho-Man and Le, Thinh T and Park, Jinyoung and Nguyen, Thanh D and Lo, Kevin W-H
Journal: Plos one (2024): e0299579
A High-Protein Diet Promotes Atrial Arrhythmogenesis via Absent-in-Melanoma 2 Inflammasome
Authors: Song, Jia and Wu, Jiao and Robichaux, Dexter J and Li, Tingting and Wang, Shuyue and Arredondo Sancristobal, Maria J and Dong, Bingning and Dobrev, Dobromir and Karch, Jason and Thomas, Sandhya S and others,
Journal: Cells (2024): 108
Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes
Authors: Oropeza-Almaz{\'a}n, Yuriana and Blatter, Lothar A
Journal: Biomolecules (2024): 144
Creating cell-specific computational models of stem cell-derived cardiomyocytes using optical experiments
Authors: Yang, Janice and Daily, Neil and Pullinger, Taylor K and Wakatsuki, Tetsuro and Sobie, Eric A
Journal: bioRxiv (2024): 2024--01
Twenty years of islet-on-a-chip: microfluidic tools for dissecting islet metabolism and function
Authors: Regeenes, Romario and Rocheleau, Jonathan V
Journal: Lab on a Chip (2024)
Non-Faradaic optoelectrodes for safe electrical neuromodulation
Authors: Chen, Jian and Liu, Yanyan and Chen, Feixiang and Guo, Mengnan and Zhou, Jiajia and Fu, Pengfei and Zhang, Xin and Wang, Xueli and Wang, He and Hua, Wei and others,
Journal: Nature Communications (2024): 405
Monolithic silicon for high spatiotemporal translational photostimulation
Authors: Li, Pengju and Zhang, Jing and Hayashi, Hidenori and Yue, Jiping and Li, Wen and Yang, Chuanwang and Sun, Changxu and Shi, Jiuyun and Huberman-Shlaes, Judah and Hibino, Narutoshi and others,
Journal: Nature (2024): 1--9
Puerarin Ameliorated PCOS through Preventing Mitochondrial Dysfunction Dependent on the Maintenance of Intracellular Calcium Homeostasis
Authors: Wang, Yu-Si and Li, Bai-Yu and Xing, Yin-Fei and Huang, Ji-Cheng and Chen, Zhi-Song and Yue, Liang and Zou, Ying-Gang and Guo, Bin
Journal: Journal of Agricultural and Food Chemistry (2024)
A PACAP-activated network for secretion requires coordination of calcium influx and calcium mobilization
Authors: Chen, Xiaohuan and Bell, Nicole A and Coffman, Breanna L and Rabino, Agustin and Garcia-Mata, Rafael and Yule, David I and Axelrod, Daniel and Smrcka, Alan and Giovannucci, David R and Anantharam, Arun
Journal: bioRxiv (2024): 2024--01
Tripeptidyl peptidase II coordinates the homeostasis of calcium and lipids in the central nervous system and its depletion causes presenile dementia in female mice through calcium/lipid dyshomeostasis-induced autophagic degradation of CYP19A1
Authors: Zhao, Jin and He, Chengtong and Fan, Xueyu and Wang, Lin and Zhao, Liao and Liu, Hui and Shen, Wujun and Jiang, Sanwei and Pei, Kaixuan and Gao, Jingjing and others,
Journal: Theranostics (2024): 1390

References


View all 72 references: Citation Explorer
Measurement and simulation of myoplasmic calcium transients in mouse slow-twitch muscle fibres
Authors: Hollingworth S, Kim MM, Baylor SM.
Journal: J Physiol (2012): 575
Mononucleated and binucleated cardiomyocytes in left atrium and pulmonary vein have different electrical activity and calcium dynamics
Authors: Huang CF, Chen YC, Yeh HI, Chen SA.
Journal: Prog Biophys Mol Biol (2012): 64
A near-infrared fluorescent calcium probe: a new tool for intracellular multicolour Ca2+ imaging
Authors: Matsui A, Umezawa K, Shindo Y, Fujii T, Citterio D, Oka K, Suzuki K.
Journal: Chem Commun (Camb) (2011): 10407
Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi
Authors: Nair R, Raina S, Keshavarz T, Kerrigan MJ.
Journal: Fungal Biol (2011): 326
Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels
Authors: Heusinkveld HJ, Westerink RH.
Journal: Toxicol Appl Pharmacol (2011): 1
Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling
Authors: Longden TA, Dunn KM, Draheim HJ, Nelson MT, Weston AH, Edwards G.
Journal: Br J Pharmacol (2011): 922
Nanoneedle transistor-based sensors for the selective detection of intracellular calcium ions
Authors: Son D, Park SY, Kim B, Koh JT, Kim TH, An S, Jang D, Kim GT, Jhe W, Hong S.
Journal: ACS Nano (2011): 3888
Ethanol alters calcium signaling in axonal growth cones
Authors: Mah SJ, Fleck MW, Lindsley TA.
Journal: Neuroscience (2011): 384
Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers
Authors: Olojo RO, Hern and ez-Ochoa EO, Ikemoto N, Schneider MF.
Journal: Biophys J (2011): 2367
Dextran-coated silica nanoparticles for calcium-sensing
Authors: Schulz A, Woolley R, Tabarin T, McDonagh C.
Journal: Analyst (2011): 1722