Rhod-5N, AM
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-5N AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Rhod-5N AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Rhod-5N 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-5N 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-5N 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-5N 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 | 86.586 µL | 432.93 µL | 865.861 µL | 4.329 mL | 8.659 mL |
5 mM | 17.317 µL | 86.586 µL | 173.172 µL | 865.861 µL | 1.732 mL |
10 mM | 8.659 µL | 43.293 µL | 86.586 µL | 432.93 µL | 865.861 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Fluo-5N, AM *Cell permeant* | 494 | 516 | - |
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-4™, AM | 523 | 551 | 0.11 |
Rhod-FF, AM | 553 | 577 | - |
Citations
Authors: Luo, Dun and Chen, Qian and Xiao, Zhuojie and Feng, Cong and Hu, Ruitao and Wang, Yuyi and Zhu, Ce and Yang, Xi and Liu, Limin and Li, Xiangfeng and others,
Journal: Regenerative Biomaterials (2025): rbaf069
Authors: Zhang, Yunxia and Wu, Qiqian and Bai, Furong and Hu, Yanqin and Xu, Bufang and Tang, Yujie and Wu, Jingwen
Journal: Journal of Ovarian Research (2025): 75
Authors: Wu, Lina and Wang, Yingfei and He, Rong and Zhang, Yue and He, Yuling and Wang, Chao and Lu, Zhenda and Liu, Ying and Ju, Huangxian
Journal: Analytica Chimica Acta (2019)
Authors: Miyake, Takahito and Shirakawa, Hisashi and Nakagawa, Takayuki and Kaneko, Shuji
Journal: Glia (2015): 1870--1882
Authors: G{\'o}mez Sucerquia, Leysa Jackeline
Journal: (2013)
References
Authors: Gangidi RR, Metzger LE.
Journal: J Dairy Sci (2006): 4105
Authors: McNamara CJ, Perry TDt, Bearce K, Hern and ez-Duque G, Mitchell R.
Journal: J Microbiol Methods (2005): 245