Rhod-4™, AM
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_wwHLs.jpg&w=640&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_wwHLs.jpg&w=640&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_wwHLs.jpg&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_HGqJh.jpg&w=96&q=25)
![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=128&q=25)
![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. †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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_LOQes.jpg&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Frhod-4-am%2Ffigure-for-rhod-4-am_s7uYQ.jpg&w=128&q=25)
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.
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 | = |
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 | - |
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