AAT Bioquest

Rhod-5N, AM

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Physical properties
Dissociation constant (Kd, nM)300000
Molecular weight1154.92
Spectral properties
Excitation (nm)557
Emission (nm)580
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


Molecular weight
Dissociation constant (Kd, nM)
Excitation (nm)
Emission (nm)
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-5N has a lower binding affinity for Ca2+ (Kd = ~320 µM) than any other BAPTA-based indicator and is suitable for Ca2+ measurements from 10 µM to 1 mM. Like the parent Rhod-2 indicator, Rhod-5N is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+. Rhod-5N AM is cell-permeable version of Rhod-5N.


Fluorescence microscope

ExcitationTRITC filter set
EmissionTRITC filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


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.

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


Rhod-5N AM Working Solution
On the day of the experiment, either dissolve Rhod-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, Rhod-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 Rhod-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.


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 Rhod-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.
  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 1 hour 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 TRITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm. 


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Rhod-5N, AM 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 mM86.586 µL432.93 µL865.861 µL4.329 mL8.659 mL
5 mM17.317 µL86.586 µL173.172 µL865.861 µL1.732 mL
10 mM8.659 µL43.293 µL86.586 µL432.93 µL865.861 µL

Molarity calculator

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Spectral properties

Excitation (nm)557
Emission (nm)580

Product Family

NameExcitation (nm)Emission (nm)Quantum yield
Rhod-2, AM *CAS#: 145037-81-6*5535770.11
Rhod-2, AM *UltraPure Grade* *CAS#: 145037-81-6*5535770.11
Rhod-FF, AM553577-
Rhod-4™, AM5235510.11
Fluo-5N, AM *Cell permeant*494516-



View all 4 citations: Citation Explorer
Fluorescence hydrogel array based on interfacial cation exchange amplification for highly sensitive microRNA detection
Authors: Wu, Lina and Wang, Yingfei and He, Rong and Zhang, Yue and He, Yuling and Wang, Chao and Lu, Zhenda and Liu, Ying and Ju, Huangxian
Journal: Analytica Chimica Acta (2019)
Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration
Authors: Miyake, Takahito and Shirakawa, Hisashi and Nakagawa, Takayuki and Kaneko, Shuji
Journal: Glia (2015): 1870--1882
Papel central de la mitocondria en la hepatotoxicidad inducida por Efavirenz
Authors: G{\'o}mez Sucerquia, Leysa Jackeline
Journal: (2013)
ER stress in human hepatic cells treated with Efavirenz: mitochondria again
Authors: Apostolova, Nadezda and Gomez-Sucerquia, Leysa J and Alegre, Fern and o , undefined and Funes, Haryes A and Victor, Victor M and Barrachina, Maria D and Blas-Garcia, Ana and Esplugues, Juan V
Journal: Journal of hepatology (2013): 780--789


View all 2 references: Citation Explorer
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
Measurement of limestone biodeterioration using the Ca2+ binding fluorochrome Rhod-5N
Authors: McNamara CJ, Perry TDt, Bearce K, Hern and ez-Duque G, Mitchell R.
Journal: J Microbiol Methods (2005): 245