AAT Bioquest

Rhod-FF, AM

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Physical properties
Dissociation constant (Kd, nM)19000
Molecular weight1145.90
Spectral properties
Excitation (nm)553
Emission (nm)577
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-FF AM is cell-permeable, and generates Rhod-FF upon esterase hydrolysis in cells. Rhod-FF has a lower binding affinity for Ca2+ and is suitable for Ca2+ measurements from 10 to 200 uM. Like the parent Rhod-2 indicator, Rhod-FF is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+.


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


Rhod-FF AM Working Solution
  1. On the day of the experiment, either dissolve Rhod-FF AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Rhod-FF 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, Rhod-FF 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-FF 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.


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-FF 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 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-FF, 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 mM87.268 µL436.338 µL872.676 µL4.363 mL8.727 mL
5 mM17.454 µL87.268 µL174.535 µL872.676 µL1.745 mL
10 mM8.727 µL43.634 µL87.268 µL436.338 µL872.676 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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Spectral properties

Excitation (nm)553
Emission (nm)577

Product Family



View all 2 citations: Citation Explorer
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
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