Fluo-5N, AM Cell permeant

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Dissociation constant (K_d, nM)	90000
Molecular weight	1127.92
Solvent	DMSO

Spectral properties

Excitation (nm)	494
Emission (nm)	516

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

1127.92

Dissociation constant (K_d, 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

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Fluorescence microscope

Excitation	FITC
Emission	FITC
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	490
Emission	525
Cutoff	515
Recommended plate	Black 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

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

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. 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-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 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.

Prepare cells in growth medium overnight.
On the next day, add 1X Fluo-5N 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.
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.
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.
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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	88.659 µL	443.294 µL	886.588 µL	4.433 mL	8.866 mL
5 mM	17.732 µL	88.659 µL	177.318 µL	886.588 µL	1.773 mL
10 mM	8.866 µL	44.329 µL	88.659 µL	443.294 µL	886.588 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Excitation (nm)	494
Emission (nm)	516

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	82000	0.16¹
Fluo-3, AM CAS 121714-22-5	506	515	86,000¹	0.15¹
Fluo-3, AM UltraPure grade CAS 121714-22-5	506	515	86,000¹	0.15¹
Fluo-3, AM Bulk package CAS 121714-22-5	506	515	86,000¹	0.15¹
Fluo-3FF, AM UltraPure grade Cell permeant	506	515	86,000¹	0.15¹
Fluo-8®, AM	495	516	23430	0.16¹
Fluo-8H™, AM	495	516	23430	0.16¹
Fluo-8L™, AM	495	516	23430	0.16¹
Fluo-8FF™, AM	495	516	23430	0.16¹
Fluo-5F, AM Cell permeant	494	516	-	-
Rhod-5N, AM	557	580	-	-
Show More (2)

Images

Figure 1. Chemical structure for Fluo-5N, AM *Cell permeant*

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

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