Rhod-2, AM *CAS#: 12978-64-0*
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Calcium GPCR Assays
pH and Ion Indicators
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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.
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.
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