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AAT Bioquest

Fluo-5N, AM *Cell permeant*

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.

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.

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

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--
Show More (2)

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-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

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
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)
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
Page updated on December 6, 2024

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Physical properties

Dissociation constant (Kd, nM)90000

Molecular weight

1127.92

Solvent

DMSO

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

Storage

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

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