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Cal-590™ AM

Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-2 is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Cal-590™ has been developed to improve Rhod-2 cell loading and calcium response while maintaining the spectral wavelength of Rhod-2, making it compatible with TRITC/Cy3® filter set. In CHO and HEK cells Cal-590™ AM has cellular calcium response that is much more sensitive than Rhod-2 AM. The spectra of Cal-590 is well separated from those of FITC, Alexa Fluor® 488 and GFP, making it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies.

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-590™ AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Cal-590™ AM in anhydrous DMSO.

    Note: When reconstituted in DMSO, Cal-590™ AM is a clear, colorless solution.

PREPARATION OF WORKING SOLUTION

Cal-590™ AM Working Solution
  1. On the day of the experiment, either dissolve Cal-590™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Cal-590™ 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-590™ 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-590™ 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-590™ 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 30 to 60 minutes.

    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 TRITC/Cy3 filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cal-590™ 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 mM78.938 µL394.692 µL789.384 µL3.947 mL7.894 mL
5 mM15.788 µL78.938 µL157.877 µL789.384 µL1.579 mL
10 mM7.894 µL39.469 µL78.938 µL394.692 µL789.384 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

Product family

NameExcitation (nm)Emission (nm)Quantum yield
Cal-630™ AM6096260.371
Cal-520®, AM4925150.751
Cal-520FF™, AM4925150.751
Cal-520N™, AM4925150.751
Calbryte™ 590 AM581593-
Cal-500™ AM3884820.481
Cal-520ER™ AM492515-

Citations

View all 74 citations: Citation Explorer
Dendritic, delayed, stochastic CaMKII activation in behavioural time scale plasticity
Authors: Jain, Anant and Nakahata, Yoshihisa and Pancani, Tristano and Watabe, Tetsuya and Rusina, Polina and South, Kelly and Adachi, Kengo and Yan, Long and Simorowski, Noriko and Furukawa, Hiro and others,
Journal: Nature (2024): 1--9
Computational analysis of $\beta$ cell Ca2+ dynamics in situ
Authors: Yong, Fiona Su Wern
Journal: (2024)
ER and SOCE Ca2+ signals are not required for directed cell migration in human iPSC-derived microglia
Authors: Granzotto, Alberto and McQuade, Amanda and Chadarevian, Jean Paul and Davtyan, Hayk and Sensi, Stefano L and Parker, Ian and Blurton-Jones, Mathew and Smith, Ian F
Journal: Cell Calcium (2024): 102923
Folate as a potential treatment for lethal ventricular arrhythmias in TANGO2-deficiency disorder
Authors: Xu, Weiyi and Cao, Yingqiong and Stephens, Sara B and Arredondo, Maria Jose and Chen, Yifan and Perez, William and Sun, Liang and Andy, C Yu and Kim, Jean J and Lalani, Seema R and others,
Journal: JCI Insight (2024)
Page updated on November 3, 2024

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10x50 ug
1 mg
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Physical properties

Dissociation constant (Kd, nM)561

Molecular weight

1266.81

Solvent

DMSO

Spectral properties

Extinction coefficient (cm -1 M -1)

78000

Excitation (nm)

574

Emission (nm)

588

Quantum yield

0.621

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationTRITC, Cy3
EmissionTRITC, Cy3
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation540
Emission590
Cutoff570
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling
ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590&reg; AM  or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 &micro;L per well in a 96-well black wall/clear bottom Costar plate. 100 &micro;L of 5 &micro;g/mL Cal-590&reg; AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 &deg;C for 1 hour. ATP (50 &micro;L/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.
ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590&reg; AM  or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 &micro;L per well in a 96-well black wall/clear bottom Costar plate. 100 &micro;L of 5 &micro;g/mL Cal-590&reg; AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 &deg;C for 1 hour. ATP (50 &micro;L/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.
ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590&reg; AM  or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 &micro;L per well in a 96-well black wall/clear bottom Costar plate. 100 &micro;L of 5 &micro;g/mL Cal-590&reg; AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 &deg;C for 1 hour. ATP (50 &micro;L/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.
Staining of astrocytes with Cal-590 in the in vivo mouse cortex. (A) Two-photon fluorescence image (average of 1,200 consecutive frames) obtained in layer 2/3 of the visual cortex of an anesthetized mouse after bulk-loading with Cal-590 AM. The excitation wavelength was 1,050 nm. (B) Two-photon fluorescence image of the same optical section as in A after additional staining with sulforhodamine 101 (SR 101). For staining with SR 101, we used the protocol initially published by Nimmerjahn et al.. The excitation wavelength was 900 nm. The red arrows here and in A indicate two labeled astrocytes. Source: <b>Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator</b> by Tischbirek, C., Birkner, A., Jia, H., Sakmann, B. & Konnerth, A. <em>Proc. Natl. Acad. Sci.</em> USA 112, 11377–11382. July 2015