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Fluo-5N, AM *Cell permeant*

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Physical properties
Dissociation constant (Kd, nM)90000
Molecular weight1127.92
SolventDMSO
Spectral properties
Excitation (nm)494
Emission (nm)516
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Molecular weight
1127.92
Dissociation constant (Kd, nM)
90000
Excitation (nm)
494
Emission (nm)
516
Fluo-5N is an analog of Fluo-4 with lower calcium-binding affinity (Kd = ~90 uM), making it suitable for detecting intracellular calcium levels in the range of 1 µM to 1 mM that would saturate the response of Fluo-4. Fluo-5N AM ester may be directly loaded into live cells by adding the dissolved indicator directly to dishes containing the cultured cells. It is compatible with excitation at 488 nm by argon-ion laser sources, making Fluo-5N useful for confocal microscopy, flow cytometry, and microplate screening applications. It has excitation and emission wavelengths at 494 and 516 nm respectively. Upon calcium binding, its fluorescence intensity increases by >100 fold.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

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

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

Fluo-5N AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Fluo-5N AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fluo-5N AM Working Solution
  1. On the day of the experiment, either dissolve Fluo-5N AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Fluo-5N 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, Fluo-5N 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 Fluo-5N 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 Fluo-5N 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 2 hours 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 490/525 nm cutoff 515 nm.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluo-5N, AM *Cell permeant* 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 mM88.659 µL443.294 µL886.588 µL4.433 mL8.866 mL
5 mM17.732 µL88.659 µL177.318 µL886.588 µL1.773 mL
10 mM8.866 µL44.329 µL88.659 µL443.294 µL886.588 µL

Molarity calculator

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

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Spectrum


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spectrum

Spectral properties

Excitation (nm)494
Emission (nm)516

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*495528820000.161
Fluo-3, AM *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *Bulk package* *CAS 121714-22-5*50651586,00010.151
Fluo-3FF, AM *UltraPure grade* *Cell permeant*50651586,00010.151
Fluo-8®, AM495516234300.161
Fluo-8H™, AM495516234300.161
Fluo-8L™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-5F, AM *Cell permeant*494516--
Rhod-5N, AM557580--
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Images


Citations


View all 13 citations: Citation Explorer
Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs
Authors: Kern, David M and Sorum, Ben and Mali, Sonali S and Hoel, Christopher M and Sridharan, Savitha and Remis, Jonathan P and Toso, Daniel B and Kotecha, Abhay and Bautista, Diana M and Brohawn, Stephen G
Journal: Nature Structural \& Molecular Biology (2021): 1--10
Involvement of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in mPR$\alpha$ (PAQR7)-mediated progesterone induction of vascular smooth muscle relaxation
Authors: Pang, Yefei and Thomas, Peter
Journal: American Journal of Physiology-Endocrinology and Metabolism (2021): E453--E466
Calreticulin regulates TGF-&beta;1-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 &amp; 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
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: Sun, Xia and Lin, Yi and Huang, Qiansheng and Shi, Junpeng and Qiu, Ling and Kang, Mei and Chen, Yajie and Fang, Chao and Ye, Ting and Dong, Sijun
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: Zhao, Lantao and Li, Shuhong and Wang, Shilei and Yu, Ning and Liu, Jia
Journal: Biochemical and biophysical research communications (2015): 537--542
Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats
Authors: Liang, Nan and Wang, Peng and Wang, Shilei and Li, Shuhong and Li, Yu and Wang, Jinying and Wang, Min
Journal: Journal of Neural Transmission (2014): 593--600
Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells
Authors: Li, Qing and Lu, Jiang and Wang, Xianyu and others, undefined
Journal: Neural regeneration research (2014): 2002

References


View all 64 references: Citation Explorer
Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure
Authors: Lu FH, Fu SB, Leng X, Zhang X, Dong S, Zhao YJ, Ren H, Li H, Zhong X, Xu CQ, Zhang WH.
Journal: Cell Physiol Biochem (2013): 728
Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle
Authors: Manno C, Sztretye M, Figueroa L, Allen PD, Rios E.
Journal: J Physiol (2013): 423
Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter
Authors: Schwartz J, Holmuhamedov E, Zhang X, Lovelace GL, Smith CD, Lemasters JJ.
Journal: Toxicol Appl Pharmacol (2013): 172
Facilitation of cytosolic calcium wave propagation by local calcium uptake into the sarcoplasmic reticulum in cardiac myocytes
Authors: Maxwell JT, Blatter LA.
Journal: J Physiol (2012): 6037
Permeation of calcium through purified connexin 26 hemichannels
Authors: Fiori MC, Figueroa V, Zoghbi ME, Saez JC, Reuss L, Altenberg GA.
Journal: J Biol Chem (2012): 40826
Post-conditioning protecting rat cardiomyocytes from apoptosis via attenuating calcium-sensing receptor-induced endo(sarco)plasmic reticulum stress
Authors: Gan R, Hu G, Zhao Y, Li H, Jin Z, Ren H, Dong S, Zhong X, Yang B, Xu C, Lu F, Zhang W.
Journal: Mol Cell Biochem (2012): 123
Fluorescence-based measurement of store-operated calcium entry in live cells: from cultured cancer cell to skeletal muscle fiber
Authors: Pan Z, Zhao X, Brotto M.
Journal: J Vis Exp. (2012)
Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation
Authors: Lu FH, Tian Z, Zhang WH, Zhao YJ, Li HL, Ren H, Zheng HS, Liu C, Hu GX, Tian Y, Yang BF, Wang R, Xu CQ.
Journal: J Biomed Sci (2010): 50
Ionic calcium determination in skim milk with molecular probes and front-face fluorescence spectroscopy: simple linear regression
Authors: Gangidi RR, Metzger LE.
Journal: J Dairy Sci (2006): 4105
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