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Rhod-4™, sodium salt

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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.
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Unit Size: Cat No: Price (USD): Qty:
21118 $495


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Overview

Ex/Em (nm)524/551
MW815.64
SolventWater
Storage F/D/L
Category GPCR
Calcium GPCR Assays
Related Calcium Channels
pH and Ion Indicators
Biochemical Assays
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.




Calculators
Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of Rhod-4™, sodium salt to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Wavelength (nm)





Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.

Use of Rhod-4™ AM Esters

1. Load Cell with Quest Rhod-4™ AM Esters:

AM esters are the non-polar esters that readily cross live cell membranes, and rapidly hydrolyzed by cellular esterases inside live cells. AM esters are widely used for loading a variety of polar fluorescent probes into live cell non-invasively. However, cautions must be excised when AM esters are used since they are susceptible to hydrolysis, particularly in solution. They should be reconstituted just before use in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions may be stored desiccated at –20 °C and protected from light. Under these conditions, AM esters should be stable for several months.

Following is our recommended protocol for loading Rhod-4™ AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.

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

b)       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 working solution of 1 to 10 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.02% Pluronic® F-127. For most of cell lines, Rhod-4™ AM reagents with a concentration ranging from 4-5 uM are recommended. The exact concentration of the indicator required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength.

Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Rhod-4™ AM esters.  A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.

c)       If your cells containing the organic anion-transports, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators.

Note: A variety of ReadiUse™ probenecid including water soluble sodium salt and stabilized solution can be purchased from AAT Bioquest.

d)       Add equal volume of the dye working solution (from Step b or c) into your cell plate.

e)       Incubate the dye-loading plate at a cell incubator or room temperature for 30 minutes to one hour at room temperature or 37 °C.

Note: Decreasing the loading temperature might reduce the compartmentalization of the indictor.

f)        Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 2.5 mM probenecid, if applicable) to remove excess probes.

g)       Run the experiments at Ex/Em  = 540/590 nm

 

2. Measure Intracellular Calcium Responses: see figure 3.

To determine either the free calcium concentration of a solution or the Kd of a single-wavelength calcium indicator, the following equation is used:

[Ca]free = Kd[F ─ Fmin]/Fmax ─ F]

Where F is the fluorescence of the indicator at experimental calcium levels, Fmin is the fluorescence in the absence of calcium and Fmax is the fluorescence of the calcium-saturated probe.

 

The dissociation constant (Kd) is a measure of the affinity of the probe for calcium. The Ca-binding and spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration solutions. In situ response calibrations of intracellular indicators typically yield Kd values significantly higher than in vitro determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca2+ buffers in the presence of ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin or Triton® X-100 can be used to expose the indicator to the controlled Ca2+ levels of the extracellular medium. The Kd value of Quest Rhod-4™ is listed in Table 1 for your reference.

 

Use of Screen Quest™ Rhod-4 NW Calcium Assay Kits for HTS Applications

GPCR activation can be detected by direct measurement of the receptor mediated cAMP accumulation, or changes in intracellular Ca2+ concentration. GPCR targets that couple via Gq produce an increase in intracellular Ca2+ that can be measured using a combination of Fluo-8® reagents and a fluorescence microplate reader. The fluorescence imaging plate readers (such as, FLIPR™, FDSS or BMG NovoStar™) have a cooled CCD camera imaging system which collects the signal from each well of a microplate (both 96 and 384-well) simultaneously. These plate readers can read at sub-second intervals, which enables the kinetics of the response to be captured, and has an integrated pipettor that may be programmed for successive liquid additions. Besides their robust applications for GPCR targets, our Screen Quest™ Rhod-4 Calcium Assay Kits can be also used for characterizing calcium ion channels and screening calcium ion channel-targeted compounds.



Rhod-4 NW

EC50 = 1.2 uM



Rhod-2 AM

EC50 = 2 uM

Figure 3. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Rhod-4 NW Assay kit and Rhod-2 AM under the same assay conditions. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100 µL of the Screen Quest™ Rhod-4 NW calcium assay kit or Rhod-2 AM  for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar™ (BMG LabTech) to achieve the final indicated concentrations. The EC50 of Rhod-4 NW is about 1.2 uM. The Ex/Em = 540/590 nm.

 

Our Screen Quest™ Rhod-4 Calcium Assay Kits have the following advantages for HTS applications:

  • Longer Wavelengths: multiple excitations @ 488, 514, 532 &546 nm; maximum emission @ ~555 nm.
  • No Wash Required and No Quencher Interference with Your Targets.
  • Robust Performance: enable calcium assays that are impossible with Rhod-2 AM.
  • Larger Assay Window: 10 times larger than Rhod-2 AM.

 

Use of Rhod-4™ Salts

Calcium calibration can be carried out by measuring the fluorescence intensity of the salt form (25 to 50 µM in fluorescence microplate readers) of the indicators in solutions with precisely known free Ca2+ concentrations. Calibration solutions can be used based on 30 mM MOPS EGTA Ca2+ buffer. In general, water contains trace amount of calcium ion. It is highly recommended to use 30 mM MOPS + 100 mM KCl, pH 7.2 as buffer system. One can simply make a 0 and 39 µM calcium stock solutions as listed below, and these 2 solutions are used to make a serial solution of different Ca2+ concentrations

A. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA

B. 39 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA + 10 mM CaCl2

 

To determine either the free calcium concentration of a solution or the Kd of a single-wavelength calcium indicator, the following equation is used:

[Ca]free = Kd[F ─ Fmin]/Fmax ─ F]

Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, Fmin is the fluorescence intensity in the absence of calcium and Fmax is the fluorescence intensity of the calcium-saturated probe.

 

The dissociation constant (Kd) is a measure of the affinity of the probe for calcium. The calcium-binding and spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration solutions. In situ response calibrations of intracellular indicators typically yield Kd values significantly higher than in vitro determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca2+ buffers in the presence of ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin or Triton® X-100 can be used to expose the indicator to the controlled Ca2+ levels of the extracellular medium. The Kd values of Fluo-8® reagents are listed in Table 1 for your reference.






References & Citations

Effect of stem cell niche elasticity/ECM protein on the self-beating cardiomyocyte differentiation of inducedpluripotent stem (iPS) cells at different stages
Authors: Mitsuhi Hirata, Tetsuji Yamaoka
Journal: Acta Biomaterialia (2017)

Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient
Authors: Masaya Shimojima, Shinsuke Yuasa, Chikaaki Motoda, Gakuto Yozu, Toshihiro Nagai, Shogo Ito, Mark Lachmann, Shin Kashimura, Makoto Takei, Dai Kusumoto
Journal: Scientific Reports (2017)

Preliminary findings on ultrasound modulation of the electromechanical function of human stem-cell-derived cardiomyocytes
Authors: Andrew William Chen, Aleksandra Klimas, Vesna Zderic, Ivan Suares Castellanos, Emilia Entcheva
Journal: (2017): 1--4

The role of spatial organization of Ca (2+) release sites in the generation of arrhythmogenic diastolic Ca (2+) release in myocytes from failing hearts.
Authors: Andriy E Belevych, Hsiang-Ting Ho, Ingrid M Bonilla, Radmila Terentyeva, Karsten E Schober, Dmitry Terentyev, Cynthia A Carnes, Sándor Györke
Journal: Basic research in cardiology (2017): 44

Dynamic polyrotaxane-coated surface for effective differentiation of mouse induced pluripotent stem cells into cardiomyocytes
Authors: Ji-Hun Seo, Mitsuhi Hirata, Sachiro Kakinoki, Tetsuji Yamaoka, Nobuhiko Yui
Journal: RSC Advances (2016): 35668--35676

Individual evaluation of cardiac marker expression and self-beating during cardiac differentiation of P19CL6 cells on different culture substrates
Authors: Tetsuji Yamaoka, Mitsuhi Hirata, Takaaki Dan, Atsushi Yamashita, Akihisa Otaka, Takahiko Nakaoki, Azizi Miskon, Sachiro Kakinoki, Atsushi Mahara
Journal: Journal of Biomedical Materials Research Part A (2016)

Involvement of aberrant calcium signalling in herpetic neuralgia
Authors: Rebekah A Warwick, Menachem Hanani
Journal: Experimental neurology (2016): 10--18

Multiple pathways for elevating extracellular adenosine in the rat hippocampal CA1 region characterized by adenosine sensor cells
Authors: Kunihiko Yamashiro, Yuki Fujii, Shohei Maekawa, Mitsuhiro Morita
Journal: Journal of Neurochemistry (2016)

OptoDyCE as an automated system for high-throughput all-optical dynamic cardiac electrophysiology
Authors: Aleksandra Klimas, Christina M Ambrosi, Jinzhu Yu, John C Williams, Harold Bien, Emilia Entcheva
Journal: Nature communications (2016)

The G protein-coupled receptor GPR157 regulates neuronal differentiation of radial glial progenitors through the Gq-IP3 pathway
Authors: Yutaka Takeo, Nobuhiro Kurabayashi, Minh Dang Nguyen, Kamon Sanada
Journal: Scientific reports (2016)


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