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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.
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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
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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.

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Spectrum


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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-
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Images


Citations


View all 532 citations: Citation Explorer
Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae
Authors: Hoffet, Michelle Salom{\'e} and Tomov, Nikola S and Hupp, Sabrina and Mitchell, Timothy J and Iliev, Asparouh I
Journal: Toxins (2024): 232
Statement on the toxicological properties and maximum residue levels of acetamiprid and its metabolites
Authors: EFSA (European Food Safety Authority), and Hernandez-Jerez, Antonio and Coja, Tamara and Paparella, Martin and Price, Anna and Henri, Jerome and Focks, Andreas and Louisse, Jochem and Terron, Andrea and Binaglia, Marco and others,
Journal: EFSA Journal (2024): e8759
Pacemaker Channels and the Chronotropic Response in Health and Disease
Authors: Hennis, Konstantin and Piantoni, Chiara and Biel, Martin and Fenske, Stefanie and Wahl-Schott, Christian
Journal: Circulation Research (2024): 1348--1378
Metabolism and cytotoxicity studies of the two hallucinogens 1cP-LSD and 4-AcO-DET in human liver and zebrafish larvae models using LC-HRMS/MS and a high-content screening assay
Authors: Gampfer, Tanja M and Sch{\"u}tz, Victoria and Schippers, Philip and Rasheed, Sari and Baumann, Jonas and Wagmann, Lea and Pulver, Benedikt and Westphal, Folker and Flockerzi, Veit and M{\"u}ller, Rolf and others,
Journal: Journal of Pharmaceutical and Biomedical Analysis (2024): 116187
Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes
Authors: Fetterman, K Ashley and Blancard, Malorie and Lyra-Leite, Davi M and Vanoye, Carlos G and Fonoudi, Hananeh and Jouni, Mariam and DeKeyser, Jean-Marc L and Lenny, Brian and Sapkota, Yadav and George, Alfred L and others,
Journal: Cell Reports (2024)
Endogenous mutant Huntingtin alters the corticogenesis via lowering Golgi recruiting ARF1 in cortical organoid
Authors: Liu, Yang and Chen, Xinyu and Ma, Yunlong and Song, Chenyun and Ma, Jixin and Chen, Cheng and Su, Jianzhong and Ma, Lixiang and Saiyin, Hexige
Journal: Molecular Psychiatry (2024): 1--16
Signalling switches maintain intercellular communication in the vascular endothelium
Authors: Buckley, Charlotte and Lee, Matthew D and Zhang, Xun and Wilson, Calum and McCarron, John G
Journal: British Journal of Pharmacology (2024)
Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells
Authors: Chen, Zhong and Lu, Xiaowen and Watsky, Mitchell A
Journal: Plos one (2024): e0301495
NeuroSeg-III: efficient neuron segmentation in two-photon Ca 2+ imaging data using self-supervised learning
Authors: Wu, Yukun and Xu, Zhehao and Liang, Shanshan and Wang, Lukang and Wang, Meng and Jia, Hongbo and Chen, Xiaowei and Zhao, Zhikai and Liao, Xiang
Journal: Biomedical Optics Express (2024): 2910--2925

References


View all 72 references: Citation Explorer
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Caveats and limitations of plate reader-based high-throughput kinetic measurements of intracellular calcium levels
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Journal: Toxicol Appl Pharmacol (2011): 1
Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling
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Journal: Br J Pharmacol (2011): 922
Nanoneedle transistor-based sensors for the selective detection of intracellular calcium ions
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