Cal-630™ AM
![ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-630™ AM (red curve). CHO-K1 cells were seeded overnight at 50,000 cells per 100 uL per well in a Costar black wall/clear bottom 96-well plate. 100 uL of 5 µM Cal-630 ™ AM in HHBS (with 1.0 mM probenecid) was added into the cells and incubated at 37 °C for 1 hour. ATP (50 uL/well) was added using FlexSation to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-630-am%2Ffigure-for-cal-630-am_iKwQI.jpg&w=640&q=75)
![ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-630™ AM (red curve). CHO-K1 cells were seeded overnight at 50,000 cells per 100 uL per well in a Costar black wall/clear bottom 96-well plate. 100 uL of 5 µM Cal-630 ™ AM in HHBS (with 1.0 mM probenecid) was added into the cells and incubated at 37 °C for 1 hour. ATP (50 uL/well) was added using FlexSation to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-630-am%2Ffigure-for-cal-630-am_iKwQI.jpg&w=640&q=75)
![ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-630™ AM (red curve). CHO-K1 cells were seeded overnight at 50,000 cells per 100 uL per well in a Costar black wall/clear bottom 96-well plate. 100 uL of 5 µM Cal-630 ™ AM in HHBS (with 1.0 mM probenecid) was added into the cells and incubated at 37 °C for 1 hour. ATP (50 uL/well) was added using FlexSation to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-630-am%2Ffigure-for-cal-630-am_iKwQI.jpg&w=128&q=25)
![Response of endogenous P2Y receptor to ATP in CHO-K cells detected with Cal-630 ™ AM. 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 uL of 5 uM Cal-630 ™ AM in HHBS (with 1.0 mM probenecid) was added into the cells and incubated at 37 °C for 1 hour. Images were recorded with a fluorescence microscope (Olympus IX71) before and after adding 10 uM ATP (final in the well) using Texas Red Channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-630-am%2Ffigure-for-cal-630-am_yOZTR.jpg&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-630™ AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-630™ AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Cal-630™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Cal-630™ 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-630™ 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-630™ 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-630™ 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 Texas Red filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 600/640 nm cutoff 630 nm.
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 77.949 µL | 389.745 µL | 779.49 µL | 3.897 mL | 7.795 mL |
5 mM | 15.59 µL | 77.949 µL | 155.898 µL | 779.49 µL | 1.559 mL |
10 mM | 7.795 µL | 38.975 µL | 77.949 µL | 389.745 µL | 779.49 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
/ | = | x | = |
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Cal-590™ AM | 574 | 588 | 0.621 |
Cal-520®, AM | 492 | 515 | 0.751 |
Cal-520FF™, AM | 492 | 515 | 0.751 |
Cal-520N™, AM | 492 | 515 | 0.751 |
Calbryte™ 630 AM | 607 | 624 | - |
Cal-500™ AM | 388 | 482 | 0.481 |
Cal-520ER™ AM | 492 | 515 | - |
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