logo
AAT Bioquest

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

Calbryte™-520L AM Stock Solution
  1. 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

Calbryte™-520L AM Working Solution
  1. 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. 

  2. 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.

  1. Prepare cells in growth medium overnight.
  2. 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.

  3. 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.

  4. 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.
  5. 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

Table 1. Volume of DMSO needed to reconstitute specific mass of Calbryte™-520L AM to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM114.847 µL574.237 µL1.148 mL5.742 mL11.485 mL
5 mM22.969 µL114.847 µL229.695 µL1.148 mL2.297 mL
10 mM11.485 µL57.424 µL114.847 µL574.237 µL1.148 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Product family

NameExcitation (nm)Emission (nm)Quantum yield
Calbryte™ 520 AM4935150.751
Calbryte™ 590 AM581593-
Calbryte™ 630 AM607624-
Calbryte™-520XL AM4935150.751

Citations

View all 11 citations: Citation Explorer
Design and validation of genetically encoded probes for the analysis of neuronal catecholamine and ATP co-transmission
Authors: Kim, Ba Run
Journal: (2019)
Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
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)
Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
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
Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
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
Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
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

View all 53 references: Citation Explorer
A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy
Authors: Orlicky J, Sulova Z, Dovinova I, Fiala R, Zahradnikova A, Jr., Breier A.
Journal: Gen Physiol Biophys (2004): 357
Comparison of human recombinant adenosine A2B receptor function assessed by Fluo-3-AM fluorometry and microphysiometry
Authors: Patel H, Porter RH, Palmer AM, Croucher MJ.
Journal: Br J Pharmacol (2003): 671
Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics
Authors: Loughrey CM, MacEachern KE, Cooper J, Smith GL.
Journal: Cell Calcium (2003): 1
A sensitive method for the detection of foot and mouth disease virus by in situ hybridisation using biotin-labelled oligodeoxynucleotides and tyramide signal amplification
Authors: Zhang Z, Kitching P.
Journal: J Virol Methods (2000): 187
Page updated on December 6, 2024

Ordering information

Price
Unit size
Catalog Number20640
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Dissociation constant (Kd, nM)91000

Molecular weight

870.72

Solvent

DMSO

Spectral properties

Excitation (nm)

493

Emission (nm)

515

Quantum yield

0.751

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationFITC
EmissionFITC
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling