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Calbryte™-520L AM
The intracellular calcium flux assay is a widely used method in monitoring signal transduction pathways and high throughput screening of G protein-coupled receptors (GPCRs) and calcium channel targets. Following the introduction of Fluo-3 in 1989, Fluo-4, Fluo-8, and Cal-520 were later developed with enhanced signal-to-background ratios and soon became the Ca2+ indicators of choice for confocal microscopy, flow cytometry, and high throughput screening applications. However, there are still a few caveats with Fluo-4. For example, like Fluo-3, Fluo-4 exhibits poor intracellular retention, and the use of probenecid is required to prevent the cell-loaded Fluo-4 from leaking out of cells. The use of probenecid with Fluo-4-based calcium assays compromises the assay results since probenecid is well-documented to have a variety of complicated cellular effects. Calbryte 520L, AM is a new fluorescent and cell-permeable calcium indicator. Like other dye AM cell loading, Calbryte 520L AM ester is non-fluorescent, and once it gets inside the cell, it is hydrolyzed by intracellular esterase and gets activated. The activated indicator is a polar molecule that is no longer capable of freely diffusing through the cell membrane and is essentially trapped inside cells. Calbryte 520L has a low affinity to calcium ions with a Kd ∼ 91 µM. Calbryte 520L produces a bright fluorescence signal in the presence of calcium at high concentrations. It has the identical excitation and emission wavelength as Fluo-4; thus, the same Fluo-4 assay settings can be readily applied to Calbryte 520L-based calcium assays.
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™-520L AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Calbryte™-520L AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Calbryte™-520L AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Calbryte™-520L 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™-520L 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™-520L 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™-520L 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 | 114.847 µL | 574.237 µL | 1.148 mL | 5.742 mL | 11.485 mL |
| 5 mM | 22.969 µL | 114.847 µL | 229.695 µL | 1.148 mL | 2.297 mL |
| 10 mM | 11.485 µL | 57.424 µL | 114.847 µL | 574.237 µL | 1.148 mL |
Molarity calculator
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Quantum yield |
| Calbryte™ 520 AM | 493 | 515 | 0.751 |
| Calbryte™ 590 AM | 581 | 593 | - |
| Calbryte™ 630 AM | 607 | 624 | - |
| Calbryte™-520XL AM | 493 | 515 | 0.751 |
Citations
Authors: Kim, Ba Run
Journal: (2019)
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry & Cell Biology (2017)
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
Journal: Neural Regeneration Research (2017): 945
Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781
Authors: Liu, Jia and Du, Qing and Zhu, He and Li, Yu and Liu, Maodong and Yu, Shoushui and Wang, Shilei
Journal: Int J Clin Exp Med (2017): 6861--6868
References
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Authors: Orlicky J, Sulova Z, Dovinova I, Fiala R, Zahradnikova A, Jr., Breier A.
Journal: Gen Physiol Biophys (2004): 357
Authors: Patel H, Porter RH, Palmer AM, Croucher MJ.
Journal: Br J Pharmacol (2003): 671
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
Journal: Cell Calcium (2003): 1
Authors: Zhang Z, Kitching P.
Journal: J Virol Methods (2000): 187
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