Home > Products > GPCR > Calcium GPCR Assays > Rhod-2, AM *CAS#: 12978-64-0*

Rhod-2, AM *CAS#: 12978-64-0*

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Chemical structure for Rhod-2, AM *CAS#: 12978-64-0*.
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Overview

Ex/Em (nm) 549/578
MW 1123.96
CAS # 129787-64-0
Solvent DMSO
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. The long-wavelength Rhod-2 Ca2+ indicators are valuable alternatives to Fluo-3 for experiments in cells and tissues that have high levels of autofluorescence. Rhod-2 AM is cell-permeable version of Rhod-2.




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Protocol


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

Use of Calcium indicator AM Esters

 

1. Load Cells with Calcium Indicator 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 in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions should 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 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 AM esters stock solution in high-quality, anhydrous DMSO.

b)       On the day of the experiment, either dissolve calcium indicators solid in DMSO or thaw an aliquot of the indicator stock solutions to room temperature. Prepare a working solution of 2 to 20 µM in the buffer of your choice (such as Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines we recommend the final concentration of calcium indicators be 4-5 uM. The exact concentration of indicators 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 probe concentration that can yield sufficient signal strength.

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

c)       If your cells (such as CHO cells) containing the organic anion-transports, probenecid (2–5 mM) or sulfinpyrazone (0.2–0.5 mM) may be added to the the dye working solution (final in well concentration will be 1-2.5 mM for probenecid, or 0.1 -0.25 mM for sulfinpyrazone) to reduce the 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 room at temperature or 37 °C for 20 minutes (especially Fluo-8 AM) to 2 hours, and then incubate the plate at room temperature for another 30 minutes.

Note1: Decreasing the loading temperature might reduce the compartmentalization of the indictor.

Note2: Incubate the Cal-520 AM longer than 2 hours gives better signal intensity for some cell lines.

f)        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 excess probes.

g)       Run the experiments at desired Ex/Em wavelengths (see Table 1).

 

2. Measure Intracellular Calcium Responses:

 


Figure 1. Response of endogenous P2Y receptor to ATP in CHO-M1 cells without probenecid. CHO-M1 cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Fluo-3 AM, Fluo-4 AM or Cal 520® AM in HHBS were added into the wells, and the cells were incubated at 37 °C for 2 hour. The dye loading medium were replaced with 100 µl HHBS, 50 µl of 300 µM ATP were added, and then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

A             B 

Figure 2. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-520® or Fluo-4 AM. CHO-K1cells were seeded overnight in 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µL of 5 µM Fluo-4 AM or the Cal-520® AM with (A) or without (B) 2.5 mM probenecid was added into the cells, and the cells were incubated at 37oC for 2 hours.  ATP (50µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.

 

Use of Calcium indicator Salts

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 Ca2+-binding and spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration solutions. In situ 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 some calcium reagents are listed in Table 1 for your reference.

 

Use of Calcium indicator Conjugates

 

Compared to the free ion indicator, dextran conjugates of these same indicators exhibit both reduced compartmentalization and much lower rates of dye leakage. Since the molecular weight of the dextran, net charge, degree of labeling, and nature of the dye may affect the experiment, researchers are advised to consult the primary literature for information specific to the application of interest.






References & Citations

GluR3B Ab’s induced oligodendrocyte precursor cells excitotoxicity via mitochondrial dysfunction
Authors: Yi Liu, Yan Chen, Wan Tong Du, Xiu Xiang Wu, Fu Xing Dong, Xue Bin Qu, Hong Bin Fan, Rui Qin Yao
Journal: Brain Research Bulletin (2017)

ABT737 reverses cisplatin resistance by regulating ER-mitochondria Ca2+ signal transduction in human ovarian cancer cells
Authors: CHUNYAN YU, XIANRUI JIANG, YE XU, LIANKUN SUN
Journal: International Journal of Oncology (2016): 2507--2519

BAX inhibitor-1 is a Ca2+ channel critically important for immune cell function and survival
Authors: D Lisak, T Schacht, A Gawlitza, P Albrecht, O Aktas, B Koop, M Gliem, HH Hofstetter, K Zanger, Geert Bultynck
Journal: Cell Death & Differentiation (2016): 358--368

Calcium efflux from the endoplasmic reticulum regulates cisplatin-induced apoptosis in human cervical cancer HeLa cells
Authors: Luyan Shen, Naiyan Wen, Meihui Xia, Yu Zhang, Weimin Liu, Ye Xu, Liankun Sun
Journal: Oncology letters (2016): 2411--2419

Failure of Elevating Calcium Induces Oxidative Stress Tolerance and Imparts Cisplatin Resistance in Ovarian Cancer Cells
Authors: Liwei Ma, Hongjun Wang, Chunyan Wang, Jing Su, Qi Xie, Lu Xu, Yang Yu, Shibing Liu, Songyan Li, Ye Xu
Journal: Aging and Disease (2016): 254

Cardiac tissue slices: preparation, handling, and successful optical mapping
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Journal: American Journal of Physiology-Heart and Circulatory Physiology (2015): H1112--H1125

Tolerance to endoplasmic reticulum stress mediates cisplatin resistance in human ovarian cancer cells by maintaining endoplasmic reticulum and mitochondrial homeostasis
Authors: Ye Xu, Chunyan Wang, Jing Su, Qi Xie, Liwei Ma, Linchuan Zeng, Yang Yu, Shibing Liu, Songyan Li, Zhixin Li
Journal: Oncology reports (2015): 3051--3060

Voltage and calcium dual channel optical mapping of cultured HL-1 atrial myocyte monolayer
Authors: Jiajie Yan, Justin K Thomson, Weiwei Zhao, Vladimir G Fast, Tong Ye, Xun Ai
Journal: JoVE (Journal of Visualized Experiments) (2015): e52542--e52542

Confocal imaging of intracellular calcium cycling in the intact heart
Authors: Neha Singh, Manvinder Kumar, James E Kelly, Gary L Aistrup, J Andrew Wasserstrom
Journal: Manual of Research Techniques in Cardiovascular Medicine (2014): 32--40

Critical roles of junctophilin-2 in T-tubule and excitation--contraction coupling maturation during postnatal development
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Journal: Cardiovascular research (2013): 54--62