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 | SDSProtocol |
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 | ||||
/ | = | x | = |
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
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
References
Authors: Territo PR, Heil J, Bose S, Evans FJ, Balaban RS.
Journal: Appl Spectrosc (2007): 138
Authors: Stamm C, del Nido PJ.
Journal: Thorac Cardiovasc Surg (2004): 127
Authors: Martin VV, Beierlein M, Morgan JL, Rothe A, Gee KR.
Journal: Cell Calcium (2004): 509
Authors: Bednar B, Cunningham ME, Kiss L, Cheng G, McCauley JA, Liverton NJ, Koblan KS.
Journal: J Neurosci Methods (2004): 247
Authors: Stamm C, Friehs I, Choi YH, Zurakowski D, McGowan FX, del Nido PJ.
Journal: Cardiovasc Res (2003): 695
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Biophys J (2001): 549
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Cell Calcium (2001): 217
Authors: Lannergren J, Westerblad H, Bruton JD.
Journal: J Muscle Res Cell Motil (2001): 265
Authors: MacGowan GA, Du C, Glonty V, Suhan JP, Koretsky AP, Farkas DL.
Journal: J Biomed Opt (2001): 23
Authors: Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC, Agid Y, Ruberg M.
Journal: J Comp Neurol (2000): 297
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)?
Are there any substitutes for probenecid in calcium assays?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?