logo
AAT Bioquest

Rhod-2 AM

CAS#: 145037-81-6
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. The long-wavelength Rhod-2 Ca2+ indicators are valuable alternatives to Fluo-3 for experiments in cells and tissues that have high levels of autofluorescence. Rhod-2 AM is cell-permeable version of Rhod-2.

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

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

PREPARATION OF WORKING SOLUTION

Rhod-2 AM Working Solution
  1. On the day of the experiment, either dissolve Rhod-2 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Rhod-2, AM *CAS#: 145037-81-6* 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 mM88.971 µL444.856 µL889.711 µL4.449 mL8.897 mL
5 mM17.794 µL88.971 µL177.942 µL889.711 µL1.779 mL
10 mM8.897 µL44.486 µL88.971 µL444.856 µL889.711 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Product family

Citations

View all 33 citations: Citation Explorer
Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexylphthalate (DEHP)
Authors: Swift, Luther M and Roberts, Anysja and Pressman, Jenna and Guerrelli, Devon and Allen, Samuel and Haq, Kazi T and Reisz, Julie A and D’Alessandro, Angelo and Posnack, Nikki Gillum
Journal: bioRxiv (2023): 2023--05
Alterations in Mitochondria-Associated Endoplasmic Reticulum Membranes Under Oxidative Stress in R28 Cells
Authors: Yang, Yuting and Wu, Jihong and Lu, Wei and Dai, Yiqin and Zhang, Youjia and Sun, Xinghuai
Journal: (2022)
Dynamic changes in $\beta$-cell [Ca2+] regulate NFAT activation, gene transcription, and islet gap junction communication
Authors: Miranda, Jose G and Schleicher, Wolfgang E and Wells, Kristen L and Ramirez, David G and Landgrave, Samantha P and Benninger, Richard KP
Journal: Molecular metabolism (2022): 101430
Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome
Authors: Yoon, Jin-Young and Daneshgar, Nastaran and Chu, Yi and Chen, Biyi and Hefti, Marco and Irani, Kaikobad and Song, Long-Sheng and Brenner, Charles and Abel, E Dale and London, Barry and others,
Journal: bioRxiv (2022)
Dynamic changes in $\beta$-cell electrical activity and [Ca2+] regulates NFATc3 activation and downstream gene transcription
Authors: Miranda, Jose G and Schleicher, Wolfgang E and Ramirez, David G and Landgrave, Samantha P and Benninger, Richard KP
Journal: BioRXiv (2020)

References

View all 8 references: Citation Explorer
Protein kinase C and myocardial calcium handling during ischemia and reperfusion: lessons learned using Rhod-2 spectrofluorometry
Authors: Stamm C, del Nido PJ.
Journal: Thorac Cardiovasc Surg (2004): 127
Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry
Authors: Stamm C, Friehs I, Choi YH, Zurakowski D, McGowan FX, del Nido PJ.
Journal: Cardiovasc Res (2003): 695
Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Biophys J (2001): 549
Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Cell Calcium (2001): 217
Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions
Authors: Lannergren J, Westerblad H, Bruton JD.
Journal: J Muscle Res Cell Motil (2001): 265
Page updated on September 20, 2024

Ordering information

Price
GradePure
Pure
UltraPure
Unit size
Catalog Number
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Dissociation constant (Kd, nM)570

Molecular weight

1123.96

Solvent

DMSO

Spectral properties

Excitation (nm)

553

Emission (nm)

577

Quantum yield

0.11

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

CAS

145037-81-6

Platform

Fluorescence microscope

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

Fluorescence microplate reader

Excitation540
Emission590
Cutoff570
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling
<strong>The intracellular distribution of free Ca<sup>2+</sup>&nbsp;was analyzed by laser scanning confocal microscopy.</strong><br />Intracellular free Ca<sup>2+</sup>&nbsp;was detected by red fluorescent probe dihydrorhod-2 AM (Rhod-2 AM). The nuclei were stained with DAPI (blue). The pEZ-LV203 vector harboring the eGFP reporter gene produced green fluorescent protein. Source:&nbsp;<strong>Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca<sup>2+</sup> dyshomeostasis in hepatocarcinoma PLC cells&nbsp;</strong>by&nbsp;Shi M et al., <em>PLOS,</em>&nbsp;<em>&nbsp;</em>Nov. 2017.
<strong>The intracellular distribution of free Ca<sup>2+</sup>&nbsp;was analyzed by laser scanning confocal microscopy.</strong><br />Intracellular free Ca<sup>2+</sup>&nbsp;was detected by red fluorescent probe dihydrorhod-2 AM (Rhod-2 AM). The nuclei were stained with DAPI (blue). The pEZ-LV203 vector harboring the eGFP reporter gene produced green fluorescent protein. Source:&nbsp;<strong>Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca<sup>2+</sup> dyshomeostasis in hepatocarcinoma PLC cells&nbsp;</strong>by&nbsp;Shi M et al., <em>PLOS,</em>&nbsp;<em>&nbsp;</em>Nov. 2017.
<strong>The intracellular distribution of free Ca<sup>2+</sup>&nbsp;was analyzed by laser scanning confocal microscopy.</strong><br />Intracellular free Ca<sup>2+</sup>&nbsp;was detected by red fluorescent probe dihydrorhod-2 AM (Rhod-2 AM). The nuclei were stained with DAPI (blue). The pEZ-LV203 vector harboring the eGFP reporter gene produced green fluorescent protein. Source:&nbsp;<strong>Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca<sup>2+</sup> dyshomeostasis in hepatocarcinoma PLC cells&nbsp;</strong>by&nbsp;Shi M et al., <em>PLOS,</em>&nbsp;<em>&nbsp;</em>Nov. 2017.
Gallery Image 2