Rhod-4™, AM

ATR effects on cardiomyocyte electrophysiology. (A) Action potentials in response to electrical pacing were imaged optically (using di4-ANBDQBS) and presented as mean ± SEM at each point for each group. (B) Quantified APD80 for control CM and ChR2-CM without ATR and with 1 μM ATR (highest optical excitability). Both A and B had n = 3–4 samples per experimental group. (C) Calcium transients in response to electrical pacing were imaged optically (using Rhod4-AM) and presented as mean ± SEM at each point for each group. (D) Quantified CTD80 for control CM and ChR2-CM at different ATR supplements. Both C and D had n = 7–33 samples for each of the eight experimental groups. (E) Example activation maps of ChR2-CM, following point electrical stimulation at the bottom; isochrones are 10 ms apart. Scale bar is 5 mm. (F) Quantified conduction velocity from the activation maps (n = 7–14 per group). (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets. Source: <strong>Cardiac Optogenetics: Enhancement by All-trans-Retinal </strong>by Yu et al., <em>Scientific Reports</em>, Nov. 2016.

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.

Nuclear calcium transients in neonatal rat ventricular cardiomyocytes (NRVCs) exposed to hypertrophic stimuli. A. Original recording of only cytoplasmic Ca<sup>2+</sup> transients in NRVC with fluo-4. B. Line scan imaging of only cytoplasmic Ca<sup>2+</sup> transients in cardiomyocytes with fluo-4. C. Original recording of only nucleus Ca<sup>2+</sup> transients in cardiomyocytes with fluo-4. D. Line scan imaging of cytoplasmic Ca<sup>2+</sup> transients in cardiomyocytes with fluo-4. E. Original recording of cytoplasmic and nucleus Ca<sup>2+</sup> transients in NRVC with a different Ca indicator, Rhod-4. F. Line scan imaging of only cytoplasmic Ca<sup>2+</sup> transients in cardiomyocytes with Rhod-4. G. Line scan imaging of nuclear Ca<sup>2+</sup> transients in cardiomyocytes with Rhod-4. H. Average values of the effects of hypertrophic stimuli on time to F/F0 in the cytoplasm of NRVCs exposed to vehicle (control; n = 19), Ang II (n = 42), ET-1 (n = 21), or PE (n = 29). I. Average values of the effects by hypertrophic stimuli on time to F/F0 in the nucleus of NRVCs exposed to vehicle (control; n = 19), Ang II (n = 42), ET-1 (n = 21), or PE (n = 29). *P < 0.05 compared to the control. **P < 0.01 compared to the control. &dagger;P < 0.001 compared to the control. Source: <strong>Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient</strong> by Shimojima et al., <em>Scientific Reports,</em> March 2017.

Response to optical stimulation in light-sensitive cardiac syncytia. (a,b) Activation maps resulting from optical stimulation (1 Hz) of <em>in vitro</em> light-sensitive cell monolayers in the island configuration. Optical stimulus strength was at most 0.07 mW/mm<sup>2</sup> greater than the threshold irradiance required to elicit a propagating response (E<sub>e,thr</sub>). Time zero corresponds to the beginning of a 20 ms-long pulse of blue light (wavelength λ = 470 nm) applied to the 1 cm-diameter region indicated by the dashed black line in (a); spacing between isochrones is 10 ms. (c,d) Same as (a,b) but for <em>in silico</em> cell monolayers. Simulated optical stimuli were at most 0.0005 mW/mm<sup>2</sup> greater than E<sub>e,thr</sub>. Here time zero corresponds to the end of each 20 ms-long illumination pulse instead of the beginning; spacing between isochrones is 10 ms. Black-coloured locations did not activate. (e,f) Select <em>in vitro </em>calcium transients from the pixel locations 1–4 indicated in (a,b) on opposite sides of the island of ChR2-expressing donor cells (CM in GD and HEK in CD) showing the wavefront activation sequence. (g,h) Select <em>in silico</em> voltage traces (analogous to those in (e,f)) from locations 1–4. Source:<strong> Optogenetics-enabled assessment of viral gene and cell therapy for restoration of cardiac excitability</strong> by Ambrosi et al., <em>Scientific Reports</em>, Dec. 2015.

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Physical properties

Dissociation constant (K_d, nM)	451
Molecular weight	1015.96
Solvent	DMSO

Spectral properties

Excitation (nm)	523
Emission (nm)	551
Quantum yield	0.1¹

Storage, safety and handling

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

Overview SDS Protocol

Molecular weight

1015.96

Dissociation constant (K_d, nM)

451

Excitation (nm)

523

Emission (nm)

551

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. Rhod-2 is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Rhod-4™ has been developed to improve Rhod-2 cell loading and calcium response while maintaining the spectral wavelength of Rhod-2. In CHO and HEK cells Rhod-4™ AM has cellular calcium response that is 10 times more sensitive than Rhod-2 AM. AAT Bioquest offers versatile packing sizes of Quest Rhod-4 to meet your special needs, e.g., 1 mg; 10x50 µg; 20x50 µg; HTS packages with no additional packaging charge.

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-4™ AM Stock Solution

Prepare a 2 to 5 mM stock solution of Rhod-4™ AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Rhod-4™ AM 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 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-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 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-4™ 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-4™, 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	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

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	523
Emission (nm)	551
Quantum yield	0.1¹

Product Family

Name	Excitation (nm)	Emission (nm)	Quantum yield
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

Figure 1. ATP-stimulated calcium responses of endogenous P2Y receptors were measured in CHO-K1 cells with Rhod-4™ AM (Cat# 21120) and Rhod-2 AM (Cat# 21064). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were incubated with 100 µL of dye loading solution using Rhod-4™ AM (4 µM, A and B) or Rhod-2 AM (4 µM, C and D) for 1 hour in a 37 °C, 5% CO2 incubator. The staining solution was replaced with 200 µL HHBS, then the cells were imaged before (A and C) and after (B and D) ATP treatment with a fluorescence microscope (Olympus IX71) using TRITC channel.

Figure 2. ATR effects on cardiomyocyte electrophysiology. (A) Action potentials in response to electrical pacing were imaged optically (using di4-ANBDQBS) and presented as mean ± SEM at each point for each group. (B) Quantified APD80 for control CM and ChR2-CM without ATR and with 1 μM ATR (highest optical excitability). Both A and B had n = 3–4 samples per experimental group. (C) Calcium transients in response to electrical pacing were imaged optically (using Rhod4-AM) and presented as mean ± SEM at each point for each group. (D) Quantified CTD80 for control CM and ChR2-CM at different ATR supplements. Both C and D had n = 7–33 samples for each of the eight experimental groups. (E) Example activation maps of ChR2-CM, following point electrical stimulation at the bottom; isochrones are 10 ms apart. Scale bar is 5 mm. (F) Quantified conduction velocity from the activation maps (n = 7–14 per group). (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets. Source: Cardiac Optogenetics: Enhancement by All-trans-Retinal by Yu et al., Scientific Reports, Nov. 2016.

Figure 3. 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 [Ca²⁺]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: The G protein-coupled receptor GPR157 regulates neuronal differentiation of radial glial progenitors through the Gq-IP3 pathway by Takeo et al., Scientific Reports, May 2016.

Figure 4. Nuclear calcium transients in neonatal rat ventricular cardiomyocytes (NRVCs) exposed to hypertrophic stimuli. A. Original recording of only cytoplasmic Ca²⁺ transients in NRVC with fluo-4. B. Line scan imaging of only cytoplasmic Ca²⁺ transients in cardiomyocytes with fluo-4. C. Original recording of only nucleus Ca²⁺ transients in cardiomyocytes with fluo-4. D. Line scan imaging of cytoplasmic Ca²⁺ transients in cardiomyocytes with fluo-4. E. Original recording of cytoplasmic and nucleus Ca²⁺ transients in NRVC with a different Ca indicator, Rhod-4. F. Line scan imaging of only cytoplasmic Ca²⁺ transients in cardiomyocytes with Rhod-4. G. Line scan imaging of nuclear Ca²⁺ transients in cardiomyocytes with Rhod-4. H. Average values of the effects of hypertrophic stimuli on time to F/F0 in the cytoplasm of NRVCs exposed to vehicle (control; n = 19), Ang II (n = 42), ET-1 (n = 21), or PE (n = 29). I. Average values of the effects by hypertrophic stimuli on time to F/F0 in the nucleus of NRVCs exposed to vehicle (control; n = 19), Ang II (n = 42), ET-1 (n = 21), or PE (n = 29). *P < 0.05 compared to the control. **P < 0.01 compared to the control. †P < 0.001 compared to the control. Source: Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient by Shimojima et al., Scientific Reports, March 2017.

Figure 5. Response to optical stimulation in light-sensitive cardiac syncytia. (a,b) Activation maps resulting from optical stimulation (1 Hz) of in vitro light-sensitive cell monolayers in the island configuration. Optical stimulus strength was at most 0.07 mW/mm² greater than the threshold irradiance required to elicit a propagating response (E_e,thr). Time zero corresponds to the beginning of a 20 ms-long pulse of blue light (wavelength λ = 470 nm) applied to the 1 cm-diameter region indicated by the dashed black line in (a); spacing between isochrones is 10 ms. (c,d) Same as (a,b) but for in silico cell monolayers. Simulated optical stimuli were at most 0.0005 mW/mm² greater than E_e,thr. Here time zero corresponds to the end of each 20 ms-long illumination pulse instead of the beginning; spacing between isochrones is 10 ms. Black-coloured locations did not activate. (e,f) Select in vitro calcium transients from the pixel locations 1–4 indicated in (a,b) on opposite sides of the island of ChR2-expressing donor cells (CM in GD and HEK in CD) showing the wavefront activation sequence. (g,h) Select in silico voltage traces (analogous to those in (e,f)) from locations 1–4. Source: Optogenetics-enabled assessment of viral gene and cell therapy for restoration of cardiac excitability by Ambrosi et al., Scientific Reports, Dec. 2015.

Conduction properties of light-sensitive cardiac syncytia. (a,b) Activation maps during electrical stimulation (1 Hz) of <em>in vitro</em> (a) and <em>in silico</em> (b) light-sensitive cell monolayers with distribution types (I, UL, UH) and delivery mode (GD, CD). In all cases, time zero corresponds to the start of the electrical stimulus; black coloured locations did not activate. For <em>in vitro</em> cases, the asterisk (*) marks the location of the bipolar pacing electrode and the spacing between isochrones lines is 10 ms. For <em>in silico</em> cases, the dashed rectangle indicates the area where transmembrane current stimulus was applied. (c) <em>In vitro</em> calcium transients at 1 Hz electrical pacing, plotted overlaid as group mean ± SEM, for control CMs, ChR2-expressing CMs (GD-UH), and co-cultures of CMs and ChR2-expressing HEK cells (CD-UL) (n = 5 per group); no significant differences. (d) Select calcium transients from the pixel locations 1–3 indicated in ((a), GD-UL and CD-UL) showing <em>in vitro</em> wavefront propagation across the monolayer. (e) Select voltage traces (analogous to those in (c)) from the pixel locations 1–6 indicated in (b, GD-UL and CD-UL) showing <em>in silico</em> wavefront propagation and upstroke morphology. Source: <strong>Optogenetics-enabled assessment of viral gene and cell therapy for restoration of cardiac excitability</strong> by Ambrosi et al., <em>Scientific Reports,</em> Dec. 2015.

Figure 6. Conduction properties of light-sensitive cardiac syncytia. (a,b) Activation maps during electrical stimulation (1 Hz) of in vitro (a) and in silico (b) light-sensitive cell monolayers with distribution types (I, UL, UH) and delivery mode (GD, CD). In all cases, time zero corresponds to the start of the electrical stimulus; black coloured locations did not activate. For in vitro cases, the asterisk (*) marks the location of the bipolar pacing electrode and the spacing between isochrones lines is 10 ms. For in silico cases, the dashed rectangle indicates the area where transmembrane current stimulus was applied. (c) In vitro calcium transients at 1 Hz electrical pacing, plotted overlaid as group mean ± SEM, for control CMs, ChR2-expressing CMs (GD-UH), and co-cultures of CMs and ChR2-expressing HEK cells (CD-UL) (n = 5 per group); no significant differences. (d) Select calcium transients from the pixel locations 1–3 indicated in ((a), GD-UL and CD-UL) showing in vitro wavefront propagation across the monolayer. (e) Select voltage traces (analogous to those in (c)) from the pixel locations 1–6 indicated in (b, GD-UL and CD-UL) showing in silico wavefront propagation and upstroke morphology. Source: Optogenetics-enabled assessment of viral gene and cell therapy for restoration of cardiac excitability by Ambrosi et al., Scientific Reports, Dec. 2015.

Citations

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The expression and function of glutamate aspartate transporters in Bergmann glia are decreased in neuronal nitric oxide synthase-knockout mice during postnatal development
Authors: Tellios, Vasiliki and Maksoud, Matthew JE and Lu, Wei-Yang
Journal: Glia (2022)

High-content analysis and Kinetic Image Cytometry identify toxicity and epigenetic effects of HIV antiretrovirals on human iPSC-neurons and primary neural precursor cells
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

Nitric oxide attenuates microglia proliferation by sequentially facilitating calcium influx through TRPV2 channels, activating NFATC2, and increasing p21 transcription
Authors: Maksoud, Matthew JE and Tellios, Vasiliki and Lu, Wei-Yang
Journal: Cell Cycle (2021): 417--433

Loperamide Inhibits Replication of Severe Fever with Thrombocytopenia Syndrome Virus
Authors: Urata, Shuzo and Yasuda, Jiro and Iwasaki, Masaharu
Journal: Viruses (2021): 869

RGS5 Attenuates Baseline Activity of ERK1/2 and Promotes Growth Arrest of Vascular Smooth Muscle Cells
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

High-content analysis and Kinetic Image Cytometry identify toxic and epigenotoxic effects of HIV antiretrovirals on human iPSC-neurons and primary neural precursor cells
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

INTEGRATION OF ENGINEERED" SPARK-CELL" SPHEROIDS FOR OPTICAL PACING OF CARDIAC TISSUE
Authors: Chua, Christianne J and Han, Julie L and Li, Weizhen and Liu, Wei and Entcheva, Emilia
Journal: bioRxiv (2021)

An innate contribution of human nicotinic receptor polymorphisms to COPD-like lesions
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

Nitric oxide displays a biphasic effect on calcium dynamics in microglia
Authors: Maksoud, Matthew JE and Tellios, Vasiliki and Xiang, Yun-Yan and Lu, Wei-Yang
Journal: Nitric Oxide (2021): 28--39

Syncytium cell growth increases Kir2. 1 contribution in human iPSC-cardiomyocytes
Authors: Li, Weizhen and Han, Julie L and Entcheva, Emilia
Journal: American Journal of Physiology-Heart and Circulatory Physiology (2020): H1112--H1122

References

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Protein kinase C and myocardial calcium handling during ischemia and reperfusion: lessons learned using Rhod-2 spectrofluorometry
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Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry
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Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching
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Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy
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Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death
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