Calbryte™ 590 AM
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 Calbryte™ 590 AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Calbryte™ 590 AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Calbryte™ 590 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Calbryte™ 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, Calbryte™ 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 Calbryte™ 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 Calbryte™ 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 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 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
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 82.05 µL | 410.25 µL | 820.499 µL | 4.102 mL | 8.205 mL |
5 mM | 16.41 µL | 82.05 µL | 164.1 µL | 820.499 µL | 1.641 mL |
10 mM | 8.205 µL | 41.025 µL | 82.05 µL | 410.25 µL | 820.499 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
/ | = | x | = |
Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Calbryte™ 520 AM | 493 | 515 | 0.751 |
Calbryte™ 630 AM | 607 | 624 | - |
Calbryte™-520L AM | 493 | 515 | 0.751 |
Calbryte™-520XL AM | 493 | 515 | 0.751 |
Cal-590™ AM | 574 | 588 | 0.621 |
Citations
Authors: Ivanova, Adelina and Atakpa-Adaji, Peace and Rao, Shanlin and Marti-Solano, Maria and Taylor, Colin W
Journal: Molecular Cell (2024)
Authors: Wenqiang, Du and Novin, Ashkan and Liu, Yamin and Afzal, Junaid and Suhail, Yasir and Liu, Shaofei and Gavin, Nicole R and Jorgensen, Jennifer R and Morosky, Christopher M and Figueroa, Reinaldo and others,
Journal: Nature Communications (2024): 8379
Authors: Scaglioni, Dominic
Journal: (2024)
Authors: Hong, Soojung and Lee, Juhee and Kim, Yunhee and Kim, Eunjee and Shin, Kunyoo
Journal: Bioengineering \& Translational Medicine (2024): e10690
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Journal: Br J Pharmacol (2003): 671
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
Journal: Cell Calcium (2003): 1
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Journal: J Virol Methods (2000): 187