Calbryte™ 520 AM
Price | |
Catalog Number | |
Availability | In stock |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Dissociation constant (Kd, nM) | 1200 |
Molecular weight | 1090.90 |
Solvent | DMSO |
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | ![]() ![]() |
Molecular weight 1090.90 | Dissociation constant (Kd, nM) 1200 | Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
Platform
Flow cytometer
Excitation | 488 nm laser |
Emission | 530/30 nm filter |
Instrument specification(s) | FITC channel |
Fluorescence microscope
Excitation | FITC |
Emission | FITC |
Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
Excitation | 490 |
Emission | 525 |
Cutoff | 515 |
Recommended plate | Black wall/clear bottom |
Instrument specification(s) | Bottom read mode/Programmable liquid handling |
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™ 520 AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Calbryte™ 520 AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Calbryte™ 520 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Calbryte™ 520 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™ 520 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™ 520 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™ 520 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 FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 91.667 µL | 458.337 µL | 916.674 µL | 4.583 mL | 9.167 mL |
5 mM | 18.333 µL | 91.667 µL | 183.335 µL | 916.674 µL | 1.833 mL |
10 mM | 9.167 µL | 45.834 µL | 91.667 µL | 458.337 µL | 916.674 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
![spectrum](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fspectra%2Fcalbryte_520.png&w=2048&q=50)
Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Product Family
Name | Excitation (nm) | Emission (nm) |
Calbryte™ 590 AM | 581 | 593 |
Calbryte™ 630 AM | 607 | 624 |
Calbryte™-520L AM | 493 | 515 |
Calbryte™-520XL AM | 493 | 515 |
Cal-520®, AM | 492 | 515 |
Mag-520™ AM | 506 | 525 |
SoNa™ 520 AM | 491 | 511 |
Images
![ATP response was measured in CHO-K1 cells using Calbryte™ 520 AM (Cat No. 20653) and Fluo-4, AM (Cat No. 20550). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of either 10 µg/mL Calbryte™ 520 AM in HH Buffer with probenecid or 10 µg/mL Fluo-4, AM in HH Buffer with probenecid was added to the wells and incubated for 45 minutes at 37°C. Both dye loading solutions were removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added to achieve the final indicated concentration of 10 µM. Images were acquired on a Keyence microscope in the FITC channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-520-am%2Ffigure-for-calbryte-520-am_GeTxl.jpg&w=3840&q=75)
![Carbachol dose-response was measured in CHO-M1 cells with Calbryte™ 520 AM and Fluo-4 AM. CHO-M1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 520 AM in HH Buffer or 10 µg/ml Fluo-4 in HH Buffer was added and incubated for 45 minutes at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. Carbachol (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fcalbryte-520-am%2Ffigure-for-calbryte-520-am_k1JPc.jpg&w=3840&q=75)
Citations
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Journal: Biomedicine \& Pharmacotherapy (2024): 116775
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Application notes
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