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Calbryte™ 520 AM

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<p>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.</p>
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Overview

Ex/Em (nm) 492/514
MW 1090.90
CAS # N/A
Solvent DMSO
Storage F/D/L
Category Cell Biology
pH and Ion Indicators
Related
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.




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Protocol


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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

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

Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats
Authors: Nan Liang, Peng Wang, Shilei Wang, Shuhong Li, Yu Li, Jinying Wang, Min Wang
Journal: Journal of Neural Transmission (2014): 593--600

Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve--mast cell interaction in a hapten-induced mouse model of atopic dermatitis
Authors: M Hagiyama, T Inoue, T Furuno, T Iino, S Itami, M Nakanishi, H Asada, Y Hosokawa, A Ito
Journal: British Journal of Dermatology (2013): 771--778




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