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Rhod-2, AM *CAS#: 145037-81-6*

<strong>The intracellular distribution of free Ca<sup>2+</sup> was analyzed by laser scanning confocal microscopy.</strong><br>Intracellular free Ca<sup>2+</sup> 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: <strong>Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca<sup>2+</sup> dyshomeostasis in hepatocarcinoma PLC cells </strong>by Shi M et al., <em>PLOS,</em> <em> </em>Nov. 2017.
<strong>The intracellular distribution of free Ca<sup>2+</sup> was analyzed by laser scanning confocal microscopy.</strong><br>Intracellular free Ca<sup>2+</sup> 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: <strong>Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca<sup>2+</sup> dyshomeostasis in hepatocarcinoma PLC cells </strong>by Shi M et al., <em>PLOS,</em> <em> </em>Nov. 2017.
Chemical structure for Rhod-2, AM *CAS#: 145037-81-6*
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Catalog Number21060
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Physical properties
Dissociation constant (Kd, nM)570
Molecular weight1123.96
SolventDMSO
Spectral properties
Excitation (nm)553
Emission (nm)577
Quantum yield0.11
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
UNSPSC12352200

OverviewpdfSDSpdfProtocol


CAS
145037-81-6
Molecular weight
1123.96
Dissociation constant (Kd, nM)
570
Excitation (nm)
553
Emission (nm)
577
Quantum yield
0.11
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.

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

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
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
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. 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-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 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.
  1. Prepare cells in growth medium overnight.
  2. On the next day, add 1X Rhod-2 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. 

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
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)553
Emission (nm)577
Quantum yield0.11

Citations


View all 32 citations: Citation Explorer
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)
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
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)
Electrophysiological Effects of Extracellular Vesicles Secreted by Cardiosphere-Derived Cells: Unraveling the Antiarrhythmic Properties of Cell Therapies
Authors: G{\'o}mez-Cid, Lidia and Moro-L{\'o}pez, Marina and de la Nava, Ana S and Hern{\'a}ndez-Romero, Ismael and Fern{\'a}ndez, Ana I and Su{\'a}rez-Sancho, Susana and Atienza, Felipe and Grigorian-Shamagian, Lilian and Fern{\'a}ndez-Avil{\'e}s, Francisco
Journal: Processes (2020): 924
Influenza M2 protein regulates MAVS-mediated signaling pathway through interacting with MAVS and increasing ROS production
Authors: Wang, Ruifang and Zhu, Yinxing and Lin, Xian and Ren, Chenwei and Zhao, Jiachang and Wang, Fangfang and Gao, Xiaochen and Xiao, Rong and Zhao, Lianzhong and Chen, Huanchun and others,
Journal: Autophagy (2019): 1163--1181
Optocardiography and electrophysiology studies of ex vivo langendorff-perfused hearts
Authors: Swift, Luther M and Jaimes III, Rafael and McCullough, Damon and Burke, Morgan and Reilly, Marissa and Maeda, Takuya and Zhang, Hanyu and Ishibashi, Nobuyuki and Rogers, Jack M and Posnack, Nikki Gillum
Journal: Journal of visualized experiments: JoVE (2019)
Development of a deep two-photon calcium imaging method for the analysis of cortical processing in the mammalian brain
Authors: Birkner, Antje
Journal: (2019)
Kv2. 1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons
Authors: Vierra, Nicholas C and Kirmiz, Michael and van der List, Deborah and Santana, L Fernando and Trimmer, James S
Journal: Elife (2019): e49953
Bcl-2 overexpression reduces cisplatin cytotoxicity by decreasing ER-mitochondrial Ca2+ signaling in SKOV3 cells
Authors: Xu, Lu and Xie, Qi and Qi, Ling and Wang, Chunyan and Xu, Na and Liu, Weimin and Yu, Yang and Li, Songyan and Xu, Ye
Journal: Oncology reports (2018): 985--992

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
Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy
Authors: MacGowan GA, Du C, Glonty V, Suhan JP, Koretsky AP, Farkas DL.
Journal: J Biomed Opt (2001): 23
Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death
Authors: Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC, Agid Y, Ruberg M.
Journal: J Comp Neurol (2000): 297
Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2
Authors: Del Nido PJ, Glynn P, Buenaventura P, Salama G, Koretsky AP.
Journal: Am J Physiol (1998): H728