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Fluo-5F, AM *Cell permeant*

Chemical structure for Fluo-5F, AM *Cell permeant*
Chemical structure for Fluo-5F, AM *Cell permeant*
Ordering information
Price ()
Catalog Number20560
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)2300
Molecular weight1100.91
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
1100.91
Dissociation constant (Kd, nM)
2300
Excitation (nm)
494
Emission (nm)
516
Fluo-5F is an analog of Fluo-4 with lower calcium-binding affinity (Kd = ~2.3 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. Cells may be loaded with Fluo-5F AM ester by adding the dissolved indicator directly to dishes containing cultured cells. It is compatible with excitation at 488 nm by argon-ion laser sources, making Fluo-5F 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-5F AM Stock Solution
Prepare a 2 to 5 mM stock solution of Fluo-5F AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fluo-5F AM Working Solution
On the day of the experiment, either dissolve Fluo-5F AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Fluo-5F 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-5F 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 solution, 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-5F AM working solution into 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-5F, 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 mM90.834 µL454.17 µL908.339 µL4.542 mL9.083 mL
5 mM18.167 µL90.834 µL181.668 µL908.339 µL1.817 mL
10 mM9.083 µL45.417 µL90.834 µL454.17 µL908.339 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)494
Emission (nm)516

Citations


View all 11 citations: Citation Explorer
Vitamin D is an endogenous partial agonist of the transient receptor potential vanilloid 1 channel
Authors: Long, Wentong and Fatehi, Mohammad and Soni, Shubham and Panigrahi, Rashmi and Philippaert, Koenraad and Yu, Yi and Kelly, Rees and Boonen, Brett and Barr, Amy and Golec, Dominic and others,
Journal: The Journal of Physiology (2020): 4321--4338
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
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
Fungus induces the release of IL-8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways.
Authors: Peng, Xu-Dong and Zhao, Gui-Qiu and Lin, Jing and Jiang, Nan and Xu, Qiang and Zhu, Cheng-Cheng and Qu, Jain-Qiu and Cong, Lin and Li, Hui
Journal: International journal of ophthalmology (2014): 441--447

References


View all 71 references: Citation Explorer
Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles
Authors: Bian T, Autry JM, Casemore D, Li J, Thomas DD, He G, Xing C.
Journal: Biochem Biophys Res Commun (2016): 206
Single cell and subcellular measurements of intracellular Ca(2)(+) concentration
Authors: McCarron JG, Olson ML, Chalmers S, Girkin JM.
Journal: Methods Mol Biol (2013): 239
Astrocyte calcium signals at Schaffer collateral to CA1 pyramidal cell synapses correlate with the number of activated synapses but not with synaptic strength
Authors: Honsek SD, Walz C, Kafitz KW, Rose CR.
Journal: Hippocampus (2012): 29
Preferential loading of bergmann glia with synthetic acetoxymethyl calcium dyes
Authors: Hoogl, undefined and TM, Kuhn B, Wang SS.
Journal: Cold Spring Harb Protoc (2011): 1228
Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F
Authors: Eaddy AC, Schnellmann RG.
Journal: Biochem Biophys Res Commun (2011): 424
Differential sensitivity of Ca(2)+ wave and Ca(2)+ spark events to ruthenium red in isolated permeabilised rabbit cardiomyocytes
Authors: MacQuaide N, Ramay HR, Sobie EA, Smith GL.
Journal: J Physiol (2010): 4731
Reduced SERCA2 abundance decreases the propensity for Ca2+ wave development in ventricular myocytes
Authors: Stokke MK, Hougen K, Sjaastad I, Louch WE, Briston SJ, Enger UH, Andersson KB, Christensen G, Eisner DA, Sejersted OM, Trafford AW.
Journal: Cardiovasc Res (2010): 63
Regulation of postsynaptic Ca2+ influx in hippocampal CA1 pyramidal neurons via extracellular carbonic anhydrase
Authors: Fedirko N, Avshalumov M, Rice ME, Chesler M.
Journal: J Neurosci (2007): 1167
Measurement and modeling of Ca2+ waves in isolated rabbit ventricular cardiomyocytes
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