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.

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Physical properties

Dissociation constant (K_d, 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-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

Alternative formats

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

Overview SDS Protocol

CAS

145037-81-6

Molecular weight

1123.96

Dissociation constant (K_d, 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

Excitation	TRITC filter set
Emission	TRITC filter set
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	540
Emission	590
Cutoff	570
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.

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.

Prepare cells in growth medium overnight.
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.
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.
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 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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	88.971 µL	444.856 µL	889.711 µL	4.449 mL	8.897 mL
5 mM	17.794 µL	88.971 µL	177.942 µL	889.711 µL	1.779 mL
10 mM	8.897 µL	44.486 µL	88.971 µL	444.856 µL	889.711 µL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	553
Emission (nm)	577
Quantum yield	0.1¹

Product Family

Name	Excitation (nm)	Emission (nm)
Fura-2, AM CAS 108964-32-5	336	505
Fura-2, AM UltraPure Grade CAS 108964-32-5	336	505
Rhod-5N, AM	557	580
Rhod-FF, AM	553	577
Rhod-4™, AM	523	551

Images

Figure 1. The intracellular distribution of free Ca²⁺ was analyzed by laser scanning confocal microscopy.
Intracellular free Ca²⁺ 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: Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca²⁺ dyshomeostasis in hepatocarcinoma PLC cells by Shi M et al., PLOS, Nov. 2017.

Figure 2. Chemical structure for Rhod-2, AM *CAS#: 145037-81-6*

Citations

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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)

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Journal: Processes (2020): 924

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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