Cal-520®, AM
Price | |
Catalog Number | |
Availability | In stock |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Dissociation constant (Kd, nM) | 320 |
Molecular weight | 1102.95 |
Solvent | DMSO |
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Molecular weight 1102.95 | Dissociation constant (Kd, nM) 320 | Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
Platform
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
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
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.
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.
- Prepare cells in growth medium overnight.
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.
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.
- 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
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 90.666 µL | 453.33 µL | 906.659 µL | 4.533 mL | 9.067 mL |
5 mM | 18.133 µL | 90.666 µL | 181.332 µL | 906.659 µL | 1.813 mL |
10 mM | 9.067 µL | 45.333 µL | 90.666 µL | 453.33 µL | 906.659 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum

Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Product Family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Cal-520® maleimide | 493 | 515 | 0.751 |
Cal-520FF™, AM | 493 | 515 | 0.751 |
Cal-520N™, AM | 493 | 515 | 0.751 |
Cal-520® amine | 493 | 515 | 0.751 |
Cal-520® azide | 493 | 515 | 0.751 |
Cal-520® alkyne | 493 | 515 | 0.751 |
Cal-590™ AM | 574 | 588 | 0.621 |
Cal-630™ AM | 609 | 626 | 0.371 |
Calbryte™ 520 AM | 493 | 515 | 0.751 |
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Images





![Loading induced chromatin condensation is regulated by calcium signaling. (A) Representative [Ca<sup>2+</sup>]<sub>i</sub> oscillations (red arrows) in MSCs as a function of time (bar = 100 μm). Addition of ATP (B) or application of 30s DL (C) decreased the time between peeks and increased number of peaks observed in 10 min (n = ~15, *p < 0.05 vs. CM control (0% strain/0mM ATP, mean ± s.d.). (D,E) Pretreatment with BAPTA or KN62 blocked load-induced chromatin condensation, whereas pretreatment with thapsigargin (TG, F) had no effect and Verapamil (VP, G) blocked only the short term increase in CCP (at 600s of DL) (red line: CM control, green line: 600s DL, blue line: 3 h DL, n = ~20 per condition, *p < 0.05 vs. CM control, mean ± s.e.m.). (H) Schematic illustration outlining the operative signaling pathways controlling chromatin condensation with short term (600s) or long term (3h) loading (⊗ a component that is not on the critical path for load induced chromatin condensation at that time point. ATP: Adenosine triphosphate, HMCLs: hemichannels, P2YR: P2Y purinergic receptors, P2XR: P2X purinergic receptors, ER: endoplasmic reticulum, Ca: calcium. CBPs: calcium binding proteins, VGCC: voltage-gated calcium channels, TRPV4: Transient receptor potential cation channel subfamily V member 4, PIEZO: Piezo-type mechanosensitive ion channels, HDAC: Histone deacetylase, MTF: Histone methyltransferase, LICC: load induced chromatin condensation). Source: <strong>Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells</strong> by Heo et al., <em>Scientific Reports</em>, Nov. 2015.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-520-am%2Ffigure-for-cal-520-am_dwIXN.jpg&w=3840&q=75)
![CaV1.3-mediated Ca2+ signalling emerges in LNCaP during CSFBS culturing. (A) Pseudocolour micrographs of Cal-520 AM-loaded LNCaP (cultured in media containing normal FBS) during live-cell recordings. Pseudocolour scale indicating increased fluorescence intensity from blue–green–yellow–red, indicating increased intracellular [Ca2+]. Fluorescence micrographs in normal Hanks' (0 s) and during perfusion with high K+ Hanks containing Bay K 8644 (10 µM, denoted by solid horizontal bar) (100 s) are shown. Five cells (1–5, white arrows) were selected and ΔF/F0 measured then plotted on a fluorescence intensity-time graph (coloured lines, lower panel) (N = 5 recordings). (B) Pseudocolour micrographs showing CSFBS-cultured LNCaP (10 days) pre- (0 s) and during exposure (100 s) to high K+/Bay K 8644. Fluorescence intensity of 5 selected cells demonstrating Ca2+-transients in 4 of the 5 cells (lower panel) (N = 3 recordings). (C) Mean percentage of responding cells per recording is shown at several time points during CSFBS along with matched time controls. Data analysed with 1-way ANOVA with Šídák's multiple comparison test, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) (N = 3). (D) Nifedipine (1 µM) significantly reduced the percentage of high K+/Bay K 8644-responding cells after 10 day CSFBS. Data was analysed with unpaired t-test where p < 0.001 (***) (N = 5 recordings). (E) Significant knockdown of CACNA1D by siRNA transfection was achieved in CSBFS-treated LNCaP (14 days, Mann–Whitney, N = 4, p < 0.05 (*). (F) The percentage of high K+/Bay K8644-responsive LNCaP in siRNA-LNCaP was markedly reduced compared with scrambled controls, (p = 0.0578, unpaired t-test, N = 3). Source: <b>CACNA1D overexpression and voltage-gated calcium channels in prostate cancer during androgen deprivation</b> by McKerr, Niamh et.al., <em>Scientific Reports</em>, March 2023](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-520-am%2Ffigure-for-cal-520-am_Tjr1o.png&w=3840&q=75)

Citations
Authors: Chen, Feng and Dong, Xi and Wang, Zhenhuan and Wu, Tongrui and Wei, Liangpeng and Li, Yuanyuan and Zhang, Kai and Ma, Zengguang and Tian, Chao and Li, Jing and others,
Journal: Neural Regeneration Research (2024): 425--433
Authors: Zhu, Wenjia and Zheng, Yufan and Liu, Jiaying and Zhao, Chao and Sun, Ning and Qu, Xiuxia and Yang, Hui
Journal: Acta Cardiologica Sinica (2023): 580
Authors: Finkel, Samuel and Sweet, Shannon and Locke, Tyler and Smith, Sydney and Wang, Zhefan and Sandini, Christopher and Imredy, John and He, Yufang and Durante, Marc and Lagrutta, Armando and others,
Journal: APL Bioengineering (2023)
Authors: Li, Yu-Meng and Patel, Kapil D and Han, Yun-Goo and Hong, Suk-Min and Meng, Yu-Xuan and Lee, Hae-Hyoung and Park, Jeong-Hui and Knowles, Jonathan C and Hyun, Jung Keun and Lee, Jung-Hwan and others,
Journal: Chemical Engineering Journal (2023): 143125
Authors: Parker, Marina and Annamdevula, Naga S and Pleshinger, Donald and Ijaz, Zara and Jalkh, Josephine and Penn, Raymond and Deshpande, Deepak and Rich, Thomas C and Leavesley, Silas J
Journal: Bioengineering (2023): 642
Authors: Lucas-Herald, Angela K and Montezano, Augusto C and Alves-Lopes, Rheure and Haddow, Laura and O’Toole, Stuart and Flett, Martyn and Lee, Boma and Amjad, S Basith and Steven, Mairi and McNeilly, Jane and others,
Journal: The Journal of Clinical Endocrinology \& Metabolism (2023): dgad525
Authors: Uzieliene, Ilona and Bironaite, Daiva and Bagdonas, Edvardas and Pachaleva, Jolita and Sobolev, Arkadij and Tsai, Wei-Bor and Kvederas, Giedrius and Bernotiene, Eiva
Journal: International Journal of Molecular Sciences (2023): 2915
Authors: Lu, Hua-Rong and Seo, Manabu and Kreir, Mohamed and Tanaka, Tetsuya and Yamoto, Rie and Altrocchi, Cristina and van Ammel, Karel and Tekle, Fetene and Pham, Ly and Yao, Xiang and others,
Journal: Cells (2023): 958
Authors: Pungor, Judit R and Allen, V Angelique and Songco-Casey, Jeremea O and Niell, Cristopher M
Journal: bioRxiv (2023): 2023--02
Authors: Landstrom, Andrew P and Yang, Qixin and Sun, Bo and Perelli, Robin M and Bidzimou, Minu-Tshyeto and Zhang, Zhushan and Aguilar-Sanchez, Yuriana and Alsina, Katherina M and Cao, Shuyi and Reynolds, Julia O and others,
Journal: Circulation: Arrhythmia and Electrophysiology (2023): e010858
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Application notes
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Cal-520 ® , Cal-590 ™, and Cal-630™ Calcium Detection Reagents
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