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AAT Bioquest

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

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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
Certificate of OriginDownload PDF
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 *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.
  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 *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 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

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Spectrum


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spectrum

Spectral properties

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

Product Family


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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 &beta;-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)