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| Product Name | Rhod-2 AM Calcium Indicator (Cell Permeant) |
| Catalog No. | RESK-0566 |
| Description | Rhod-2 is a visible-light-excited, high-affinity Ca2+ indicator. Compared with other visible-light-excited probes such as Fluo-3 and Fluo-4, Rhod-2 offers longer wavelengths and is better suited for measuring Ca2+ levels in cells and tissues with high autofluorescence, as well as for detecting Ca2+ release triggered by photoactivation of photoreceptors and caged calcium chelators. Compared with UV-excited indicators such as Fura-2, visible-light-excited Ca2+ probes offer several advantages: 1) efficient excitation by most instruments, including confocal laser scanning microscopes and flow cytometers; 2) stronger dye absorbance, allowing successful Ca2+ detection at lower probe concentrations and reducing phototoxicity to live cells; 3) Ca2+-dependent fluorescence enhancement for higher sensitivity; 4) reduced interference from sample autofluorescence and light scattering; 5) compatibility with photoactivatable probes and UV-excited reagents for multiparameter assays; 6) no spectral shift; 7) long-wavelength emission compatible with GFP and green fluorescent probes; 8) preferential mitochondrial localization: the rhodamine-based Rhod-2 AM is positively charged and accumulates in mitochondria in a membrane-potential-driven manner, showing a punctate staining pattern by fluorescence microscopy. |
| Intended Use | Measurement of intracellular Ca2+ levels in live cells and tissues, particularly mitochondrial Ca2+, in samples with high autofluorescence, by fluorescence microscopy, confocal microscopy, or flow cytometry. |
| Principle / Technology | Rhod-2 AM is a positively charged, rhodamine-based, cell-permeant Ca2+ indicator. It crosses the cell membrane and accumulates preferentially in mitochondria via membrane-potential-driven uptake. Intracellular esterases hydrolyze the AM ester, and the resulting Rhod-2 exhibits Ca2+-dependent fluorescence enhancement upon binding Ca2+. |
| Detection Method | Red fluorescence detection (Ex/Em 549/578 nm, Ca2+-bound) by fluorescence microscopy, confocal laser scanning microscopy, or flow cytometry. |
| Sample Type | Live cells and tissues; live-cell detection is required. Suitable for samples with high autofluorescence. |
| Performance Range / Specifications | CAS No.: 129787-64-0 Molecular Formula: C52H59N4O19Br Molecular Weight: 1123.9575 Ex/Em: 549/578 nm (Ca2+-bound) |
| Sensitivity / LOD | Ca2+-dependent fluorescence enhancement provides high sensitivity for detecting changes in Ca2+ levels; strong dye absorbance permits detection at low probe concentrations. |
| Specificity | High-affinity Ca2+ indicator; preferentially signals mitochondrial Ca2+ owing to membrane-potential-driven mitochondrial accumulation. |
| Reaction Conditions / Protocol | Dilute the probe in serum-free medium, HBSS, or PBS to prepare the working solution, which can be added directly to cells. Recommended loading time is 15-60 min (30 min is a good starting point for pilot experiments); 37 °C is recommended, room temperature is also acceptable, and 4 °C is optional. For mitochondrial staining, loading at 4 °C favors mitochondrial accumulation: at 37 °C cytosolic esterases are more active and may hydrolyze the AM group before the probe reaches mitochondria, whereas at low temperature esterase activity slows and the probe can attach to mitochondria before hydrolysis. For mouse primary cardiomyocytes, a working concentration of 2.5 μM is optimal based on customer feedback; 1 μM gives a weaker signal and 5 μM shows noticeable background. Minimize the interval between probe loading and measurement (excluding stimulation time) to reduce error. |
| Components / Formulation | Rhod-2 acetoxymethyl (AM) ester, cell permeant. |
| Storage Conditions | Store at -20 °C, protected from light and kept dry. |
| Shelf Life | 1 year when stored as recommended. |
| Package Specifications | 50 μg; 5 x 50 μg; 20 x 50 μg |
| Molecular Weight | 1123.9575 |
| Shipping Conditions | Shipped at room temperature. |
| Expiration Date / Stability | Stable for 1 year when stored at -20 °C, protected from light and dry. |
| Regulatory / Compliance | For research use only. |
| Compatibility | Compatible with GFP and green fluorescent probes owing to its long-wavelength emission; compatible with photoactivatable probes and UV-excited reagents for multiparameter detection. For combined antibody detection, live-cell conditions are required and the antibody fluorophore must be spectrally distinct from Rhod-2. |
| Recommended Buffer System | Serum-free medium, HBSS, or PBS for preparing the working solution. |
| Application Notes / Precautions | For cytosolic Ca2+ measurement, Fluo-3 or Fluo-4 is recommended; for relatively concentrated mitochondrial Ca2+, Rhod-2 is recommended. Live-cell detection is required. Minimize the time from probe loading to instrument measurement. Protect from light. |
For research use only, not for clinical use.
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