Rhod-4™, AM
Price | |
Catalog Number | |
Availability | In stock |
Unit Size | |
Quantity |
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Dissociation constant (Kd, nM) | 451 |
Molecular weight | 1015.96 |
Solvent | DMSO |
Excitation (nm) | 523 |
Emission (nm) | 551 |
Quantum yield | 0.11 |
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 |
Rhod-4™, sodium salt |
Rhod-4™, potassium salt |
Overview | ![]() ![]() |
Molecular weight 1015.96 | Dissociation constant (Kd, nM) 451 | Excitation (nm) 523 | Emission (nm) 551 | Quantum yield 0.11 |
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
Prepare a 2 to 5 mM stock solution of Rhod-4™ AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Rhod-4™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Rhod-4™ 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-4™ 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-4™ 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.
- Prepare cells in growth medium overnight.
On the next day, add 1X Rhod-4™ 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.
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
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 98.429 µL | 492.145 µL | 984.291 µL | 4.921 mL | 9.843 mL |
5 mM | 19.686 µL | 98.429 µL | 196.858 µL | 984.291 µL | 1.969 mL |
10 mM | 9.843 µL | 49.215 µL | 98.429 µL | 492.145 µL | 984.291 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum

Spectral properties
Excitation (nm) | 523 |
Emission (nm) | 551 |
Quantum yield | 0.11 |
Product Family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Rhod-4™ amine | 523 | 551 | 0.11 |
Rhod-4™ azide | 523 | 551 | 0.11 |
Rhod-4™ alkyne | 523 | 551 | 0.11 |
Rhod-4™ maleimide | 523 | 551 | 0.11 |
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* | 495 | 528 | 0.161 |
Rhod-2, AM *CAS#: 145037-81-6* | 553 | 577 | 0.11 |
Rhod-2, AM *UltraPure Grade* *CAS#: 145037-81-6* | 553 | 577 | 0.11 |
Rhod-5N, AM | 557 | 580 | - |
Rhod-FF, AM | 553 | 577 | - |
Images


![GPR157 couples with Gq-class of the heterotrimeric G-proteins. (A–C) Plasmids expressing indicated protein were transfected into U-2 OS cells. Rhod-4, a fluorescent calcium indicator, were used to assess changes in [Ca<sup>2+</sup>]i. Application of Ionomycin, a calcium ionopohore, to cells after experiments confirmed almost uniform uptake of Rhod-4 in these cells. (D–F) Fluorescent intensity of Rhod4 in GFP-positive cells. Mean ± s.e.m. Data were obtained from 3 independent experiments (more than 40 cells). **p < 0.01, ***p < 0.001. Scale bar: 10 μm. Source: <strong>The G protein-coupled receptor GPR157 regulates neuronal differentiation of radial glial progenitors through the Gq-IP3 pathway </strong>by Takeo et al., <em>Scientific Reports</em>, May 2016.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_NeyL7.jpg&w=3840&q=75)



Citations
Authors: Liu, Wei and Han, Julie L and Tomek, Jakub and Bub, Gil and Entcheva, Emilia
Journal: ACS Photonics (2023)
Authors: Tellios, Vasiliki and Maksoud, Matthew JE and Lu, Wei-Yang
Journal: Glia (2022)
Authors: Smith, Alyson S and Ankam, Soneela and Farhy, Chen and Fiengo, Lorenzo and Basa, Ranor CB and Gordon, Kara L and Martin, Charles T and Terskikh, Alexey V and Jordan-Sciutto, Kelly L and Price, Jeffrey H and others,
Journal: Journal of Pharmacological and Toxicological Methods (2022): 107157
Authors: Maksoud, Matthew JE and Tellios, Vasiliki and Lu, Wei-Yang
Journal: Cell Cycle (2021): 417--433
Authors: Urata, Shuzo and Yasuda, Jiro and Iwasaki, Masaharu
Journal: Viruses (2021): 869
Authors: Demirel, Eda and Arnold, Caroline and Garg, Jaspal and J{\"a}ger, Marius Andreas and Sticht, Carsten and Li, Rui and Kuk, Hanna and Wettschureck, Nina and Hecker, Markus and Korff, Thomas
Journal: Cells (2021): 1748
Authors: Smith, Alyson S and Ankam, Soneela and Farhy, Chen and Fiengo, Lorenzo and Basa, Ranor CB and Gordon, Kara L and Martin, Charles T and Terskikh, Alexey V and Jordan-Sciutto, Kelly L and Price, Jeffrey H and others,
Journal: bioRxiv (2021): 2020--09
Authors: Chua, Christianne J and Han, Julie L and Li, Weizhen and Liu, Wei and Entcheva, Emilia
Journal: bioRxiv (2021)
Authors: Routhier, Julie and Pons, St{\'e}phanie and Freidja, Mohamed Lamine and Dalstein, V{\'e}ronique and Cutrona, J{\'e}r{\^o}me and Jonquet, Antoine and Lalun, Nathalie and M{\'e}rol, Jean-Claude and Lathrop, Mark and Stitzel, Jerry A and others,
Journal: Nature communications (2021): 1--13
Authors: Maksoud, Matthew JE and Tellios, Vasiliki and Xiang, Yun-Yan and Lu, Wei-Yang
Journal: Nitric Oxide (2021): 28--39
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Application notes
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
Quest Rhod-4™ Calcium Detection Reagents and Screen Quest™ Rhod-4 NW Calcium Assay Kits
What's A Ratiometric Indicator
A Meta-Analysis of Common Calcium Indicators
FAQ
How is intracellular calcium measured?
Are there any calcium indicators that don't require probenecid (PBC)?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?