Calbryte™ 520 AM

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80060040020010.11e-2- Fluo-4 AM- Calbryte™ 520 AM Data legend Generated with Quest Graph™ Carbachol (uM) Signal/... x 100% Hover mouse to interact
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 45min 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.
Unit Size: Cat No: Price (USD): Qty:
20650 $95

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Ex/Em (nm)492/514
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Category Cell Biology
pH and Ion Indicators
Related Calcium GPCR Assays
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. Followed by Fluo-3 being introduced in 1989, Fluo-4, Fluo-8 and Cal-520 were later developed with improved signal/background ratio, and became the widely used Ca2+ indicators for confocal microscopy, flow cytometry and high throughput screening applications. However, there are still a few severe problems with Fluo-4. For example, as for Fluo-3, in all most all the intracellular calcium assays with Fluo-4 AM, 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™ 520, AM is a new fluorescent and cell-permeable calcium indicator. Like other dye AM cell loading, Calbryte™ 520 AM ester is non-fluorescent and once gets inside cells, 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 cell membrane, essentially trapped inside cells. Upon binding Ca2+ ions, Calbryte™ 520 produces bright fluorescence signal with extremely high signal/background ratio. In addition, Calbryte™ 520 demonstrates greatly improved intracellular retention. It has the identical excitation and emission wavelength as Fluo-4, thus the same Fluo-4 assay settings can be readily applied to Calbryte™ 520-based calcium assays. Calbryte™ 520 is a new generation of fluorescent indicators for the measurement of intracellular calcium. Its greatly improved signal/background ratio and intracellular retention properties make Calbryte™ 520 AM the most robust indicator for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists in live cells.

Common stock solution preparation

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

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Mass Molecular weight Volume Concentration Moles
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This protocol only provides a guideline, and should be modified according to your specific needs.
  1. Load Cells with Calbryte™ 520, Calbryte™ 590 or Calbryte™ 630 AM Esters:
    Calbryte™ AM esters should be reconstituted just before use in anhydrous DMSO. The DMSO stock solutions may be stored (desiccated) at –20 °C and protected from light. Under these conditions, AM esters should be stable for three months. Following is our recommended protocol for loading Calbryte™ 520 AM, Calbryte™ 590 AM or Calbryte™ 630 AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.

    1. Prepare a 2 to 5 mM stock solution of Calbryte™ 520 AM, Calbryte™ 590 AM or Calbryte™ 630 AM esters in anhydrous DMSO.
    2. Dissolve Calbryte™ 520 AM, Calbryte™ 590 AM or Calbryte™ 630 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 10 to 20 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.04% Pluronic® F-127. The exact concentration of the indicator 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, Calbryte™ 590 AM or Calbryte™ 630 AM esters. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
    3. If your cells (such as CHO cells) contain organic anion-transports, 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. Probenecid might not be needed even in CHO cells with Calbryte™ 520 AM
      Note: A variety of ReadiUse™ probenecid including water soluble sodium salt and stabilized solution can be purchased from AAT Bioquest.
    4. Add equal volume of the dye working solution (from Step b or c) into your cell plate.
    5. Incubate the dye-loading plate in a cell incubator for ~60 minutes, and then incubate the plate at room temperature for another 15 minutes.
    6. Replace the dye working solution with HHBS or a buffer of your choice that contains an anion transporter inhibitor, such as 1 mM probenecid, to remove excess probes.
    7. Run the calcium tests at Ex/Em = 490/525 nm for Calbryte™ 520 AM, 540/590 nm for Calbryte™ 590 AM or 610/640 nm for Calbryte™ 630 AM.

References & Citations

Measurement of Contractility and Calcium Release in Cardiac Spheroids
Authors: Christian Zuppinger
Journal: (2019): 41--52

The Bright Calbryte 520 Enables the Probenecid-Free Intracellular Calcium Assays
Authors: Qin Zhao, Muhua Yang, Haitao Guo, Ruogu Peng, Jinfang Liao, Zhenjun Diwu
Journal: Biophysical Journal (2018): 288a

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Yanjiao Wu, Xiaoli Xu, Lunkun Ma, Qian Yi, Weichao Sun, Liling Tang
Journal: The International Journal of Biochemistry & Cell Biology (2017)

Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
Authors: Jia Liu, Qing Du, He Zhu, Yu Li, Maodong Liu, Shoushui Yu, Shilei Wang
Journal: Int J Clin Exp Med (2017): 6861--6868

Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
Authors: Jiang Lu, Xue-qin Yao, Xin Luo, Yu Wang, Sookja Kim Chung, He-xin Tang, Chi Wai Cheung, Xian-yu Wang, Chen Meng, Qing Li
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: Gang Yang, Zhenghua Xiao, Xiaomei Ren, Haiyan Long, Kunlong Ma, Hong Qian, Yingqiang Guo
Journal: Scientific Reports (2017): 41781

Di (2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection
Authors: Xia Sun, Yi Lin, Qiansheng Huang, Junpeng Shi, Ling Qiu, Mei Kang, Yajie Chen, Chao Fang, Ting Ye, Sijun Dong
Journal: Journal of cellular and molecular medicine (2015): 581--594

The effect of mitochondrial calcium uniporter on mitochondrial fission in hippocampus cells ischemia/reperfusion injury
Authors: Lantao Zhao, Shuhong Li, Shilei Wang, Ning Yu, Jia Liu
Journal: Biochemical and biophysical research communications (2015): 537--542

Fungus induces the release of IL-8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways.
Authors: Xu-Dong Peng, Gui-Qiu Zhao, Jing Lin, Nan Jiang, Qiang Xu, Cheng-Cheng Zhu, Jain-Qiu Qu, Lin Cong, Hui Li
Journal: International journal of ophthalmology (2014): 441--447

Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells
Authors: Qing Li, Jiang Lu, Xianyu Wang
Journal: Neural regeneration research (2014): 2002

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