Cal Red™ R525/650 AM
![Graph illustrates signal-to-noise ration (SNR) x 100%. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal Red R525/650. ATP (50 uL/well) was added by FlexStation3 (Molecular Devices) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Fgraph-for-cal-red-r525-650-am_CAh0y.webp&w=640&q=75)
![Graph illustrates signal-to-noise ration (SNR) x 100%. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal Red R525/650. ATP (50 uL/well) was added by FlexStation3 (Molecular Devices) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Fgraph-for-cal-red-r525-650-am_CAh0y.webp&w=640&q=75)
![Graph illustrates signal-to-noise ration (SNR) x 100%. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal Red R525/650. ATP (50 uL/well) was added by FlexStation3 (Molecular Devices) to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Fgraph-for-cal-red-r525-650-am_CAh0y.webp&w=128&q=25)
![Fluorescence emission spectra of Cal Red™ R525/650 (calcium bound).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Ffigure-for-cal-red-r525-650-am_JQBc6.png&w=128&q=25)
![ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with different Ca2+ indicators under the same conditions. ATP (50 μL/well) was added by FlexStation 3 (Molecular Devices) to achieve the final indicated concentrations. (Red: Cal Red R525/650, AM; Blue: Fura Red, AM; Green: Fura-2, AM)](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Ffigure-for-cal-red-r525-650-am_C6Otl.png&w=128&q=25)
![ATP-stimulated calcium response on CHO-K1 cells incubated with Cal Red™ R525/650, AM and Fura-Red™ AM. 10 μM ATP (final concentration in the well) was added by FlexStation 3 (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Ffigure-for-cal-red-r525-650-am_pIY6f.png&w=128&q=25)
![ATP-stimulated calcium response on CHO-K1 cells incubated with Cal Red™ R525/650, AM and Fura-Red™ AM. 10 μM ATP (final concentration in the well) was added by FlexStation 3 (Molecular Devices).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcal-red-r525-650-am%2Ffigure-for-cal-red-r525-650-am_3lYtH.png&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 Red™ R525/650 AM in anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Cal Red™ R525/650 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Cal Red™ R525/650 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 Red™ R525/650 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 Red™ R525/650 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 Red™ R525/650 AM working solution into 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 1 hour 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 a FlexStation, at Ex/Em1 = 490/525 nm cutoff 515 nm and Ex/Em2 = 490/660 nm cutoff 630 nm.
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