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

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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

Certificate of Origin	Download PDF
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 *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 *UltraPure Grade* 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 *UltraPure Grade* 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 *UltraPure Grade* *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. Chemical structure for Rhod-2, AM *UltraPure Grade* *CAS#: 145037-81-6*

Citations

View all 23 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

Hypocalcemia in sepsis: analysis of the subcellular distribution of Ca2+ in septic rats and LPS/TNF-$\alpha$-treated HUVECs
Authors: He, Wencheng and Huang, Lei and Luo, Hua and Zang, Yang and An, Youzhong and Zhang, Weixing
Journal: The Journal of Infection in Developing Countries (2020): 908--917

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)

Spatially Organized β-Cell Subpopulations Control Electrical Dynamics across Islets of Langerhans
Authors: Westacott, Matthew J and Ludin, Nurin WF and Benninger, Richard KP
Journal: Biophysical Journal (2017): 1093--1108

The Sec14-like phosphatidylinositol transfer proteins Sec14l3/SEC14L2 act as GTPase proteins to mediate Wnt/Ca2+ signaling
Authors: Gong, Bo and Shen, Weimin and Xiao, Wanghua and Meng, Yaping and Meng, Anming and Jia, Shunji
Journal: Elife (2017): e26362

Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca2+ dyshomeostasis in hepatocarcinoma PLC cells
Authors: Shi, M and Zhou, L and Zhao, L and Shang, M and He, T and Tang, Z and others, undefined
Journal: PLoS Negl Trop Dis (2017): e0006074

Biomaterial Surface Can Modify HUVEC Morphology and Inflammatory Response by Regulating MicroRNA Expression
Authors: Gu, Shuangying and Tian, Baoxiang and Chen, Weicong and Zhou, Yue
Journal: Journal of Biosciences and Medicines (2017): 8

GluR3B Ab's induced oligodendrocyte precursor cells excitotoxicity via mitochondrial dysfunction
Authors: Liu, Yi and Chen, Yan and Du, Wan Tong and Wu, Xiu Xiang and Dong, Fu Xing and Qu, Xue Bin and Fan, Hong Bin and Yao, Rui Qin
Journal: Brain Research Bulletin (2017)