Cal-590™ AM
![ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590® AM or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom Costar plate. 100 µL of 5 µg/mL Cal-590® AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-590-am%2Fgraph-for-cal-590-am_G6AWp.webp&w=640&q=75)
![ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590® AM or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom Costar plate. 100 µL of 5 µg/mL Cal-590® AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-590-am%2Fgraph-for-cal-590-am_G6AWp.webp&w=640&q=75)
![ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590® AM or Rhod-2 AM under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom Costar plate. 100 µL of 5 µg/mL Cal-590® AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-590-am%2Fgraph-for-cal-590-am_G6AWp.webp&w=128&q=25)
![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](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-590-am%2Ffigure-for-cal-590-am_rRONp.jpeg&w=128&q=25)
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-590™ AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-590™ AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
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.
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.
- Prepare cells in growth medium overnight.
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.
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.
- 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 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.
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 78.938 µL | 394.692 µL | 789.384 µL | 3.947 mL | 7.894 mL |
5 mM | 15.788 µL | 78.938 µL | 157.877 µL | 789.384 µL | 1.579 mL |
10 mM | 7.894 µL | 39.469 µL | 78.938 µL | 394.692 µL | 789.384 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
/ | = | x | = |
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Cal-630™ AM | 609 | 626 | 0.371 |
Cal-520®, AM | 492 | 515 | 0.751 |
Cal-520FF™, AM | 492 | 515 | 0.751 |
Cal-520N™, AM | 492 | 515 | 0.751 |
Calbryte™ 590 AM | 581 | 593 | - |
Cal-500™ AM | 388 | 482 | 0.481 |
Cal-520ER™ AM | 492 | 515 | - |
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