Rhod-5N, AM

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Physical properties

Dissociation constant (K_d, nM)	300000
Molecular weight	1154.92
Solvent	DMSO

Spectral properties

Excitation (nm)	557
Emission (nm)	580

Storage, safety and handling

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

Overview SDS Protocol

Molecular weight

1154.92

Dissociation constant (K_d, nM)

300000

Excitation (nm)

557

Emission (nm)

580

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. Rhod-5N has a lower binding affinity for Ca2+ (Kd = ~320 µM) than any other BAPTA-based indicator and is suitable for Ca2+ measurements from 10 µM to 1 mM. Like the parent Rhod-2 indicator, Rhod-5N is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Ca2+. Rhod-5N AM is cell-permeable version of Rhod-5N.

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-5N AM Stock Solution

Prepare a 2 to 5 mM stock solution of Rhod-5N AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Rhod-5N AM 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 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-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 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-5N 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-5N, AM 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	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

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

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Spectral properties

Excitation (nm)	557
Emission (nm)	580

Product Family

Name	Excitation (nm)	Emission (nm)	Quantum yield
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-FF, AM	553	577	-
Rhod-4™, AM	523	551	0.1¹
Fluo-5N, AM Cell permeant	494	516	-

Images

Figure 1. Chemical structure for Rhod-5N, AM

Citations

View all 4 citations: Citation Explorer

Fluorescence hydrogel array based on interfacial cation exchange amplification for highly sensitive microRNA detection
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)

Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration
Authors: Miyake, Takahito and Shirakawa, Hisashi and Nakagawa, Takayuki and Kaneko, Shuji
Journal: Glia (2015): 1870--1882

Papel central de la mitocondria en la hepatotoxicidad inducida por Efavirenz
Authors: G{\'o}mez Sucerquia, Leysa Jackeline
Journal: (2013)

ER stress in human hepatic cells treated with Efavirenz: mitochondria again
Authors: Apostolova, Nadezda and Gomez-Sucerquia, Leysa J and Alegre, Fern and o , undefined and Funes, Haryes A and Victor, Victor M and Barrachina, Maria D and Blas-Garcia, Ana and Esplugues, Juan V
Journal: Journal of hepatology (2013): 780--789

References

View all 2 references: Citation Explorer

Ionic calcium determination in skim milk with molecular probes and front-face fluorescence spectroscopy: simple linear regression
Authors: Gangidi RR, Metzger LE.
Journal: J Dairy Sci (2006): 4105

Measurement of limestone biodeterioration using the Ca2+ binding fluorochrome Rhod-5N
Authors: McNamara CJ, Perry TDt, Bearce K, Hern and ez-Duque G, Mitchell R.
Journal: J Microbiol Methods (2005): 245