Fluo-5N, AM *Cell permeant*
Ordering information
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Catalog Number | |
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
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-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 |
Related products
Overview | ![]() ![]() |
See also: Calcium Indicators, Chemical Reagents, Classic Dyes, Fluoresceins, Intracellular Ions, Physiological Probes
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
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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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 |
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
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
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)
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
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
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
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
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
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
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
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
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
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
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
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
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
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)
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
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
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
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Application notes
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
FAQ
Are there any calcium indicators that don't require probenecid (PBC)?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
How do I make an AM ester stock solution?
Are there upgraded trypan blue derivatives for cell viability testing?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
How do I make an AM ester stock solution?