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Thermo Scientific™ Rhod-3 Calcium Imaging Kit
Description
The novel Rhod-3 Calcium Imaging Kit is designed for live-cell imaging of cytosolic calcium signaling in combination with green-fluorescent dyes or proteins.The red-fluorescent Rhod-3 AM dye supplied in the Rhod-3 Calcium Imaging Kit offers significant improvements over existing Ca2+-sensing dyes. The uptake into organelles has been reduced, which translates into better cytosolic localization. Also, Rhod-3 AM exhibits a large increase in fluorescence emission upon binding to calcium, making it more sensitive than other red-fluorescent calcium dyes.
- Compared to existing red calcium dyes, Rhod-3 AM exhibits a large increase (>2.5 fold) in fluorescence upon binding Ca2+ and very low fluorescence at rest (without Ca2+ binding).
- Narrow emission enables simultaneous detection with GFP or other dyes.
- Enhanced cytosolic localization for better detection of cytosolic transients
For easy cell loading, the Rhod-3 Calcium Imaging Kit contains PowerLoad™ Concentrate. Due to the unique nature of the PowerLoad™ solution, it can be used in the presence of complete culture media, thus reducing the negative effects of replacing media or loading in serum-free media.
A proprietary, water-soluble form of probenecid (which is commonly used to inhibit cellular transport and thus reduces the baseline signal) is also supplied with the Rhod-3 Calcium Imaging Kit.
Calcium indicators are important tools in signal transduction and cell-based pharmacological screening. In cells and tissues with blue or green autofluorescence, long-wavelength (i.e., red-shifted) calcium indicators provide a means to bypass this overlapping autofluorescence. Additionally, red-shifted calcium dyes are well suited for calcium imaging experiments multiplexed with green-fluorescent protein (GFP) or other green fluorescent dyes.
The Rhod-3 AM dye (included in the Rhod-3 Calcium Imaging Kit) is an improved red-shifted rhodamine-based calcium dye with an attached AM ester group. Once inside the cell, intracellular esterases cleave the AM group, trapping the dye in the cell and rendering the dye fluorescent. As calcium binds, the fluorescence intensity of the dye is enhanced. The spectral properties of Rhod-3 AM (ex/em = ∼560/600 nm) and strong fluorescence upon calcium binding provide an excellent signal window and make it our brightest red calcium dye. Additionally, Rhod-3 AM displays a more uniform cytosolic distribution and improved signal compared to existing red calcium dyes such as Rhod-2.
Specifications
Specifications
| Color | Red |
| Content And Storage | The kit contains sufficient reagents for staining 50 coverslips based upon the protocol provided. Store kit at -20°C, desiccated and protected from light. When stored as directed, this kit is stable for 1 year. |
| Detection Method | Fluorescence |
| For Use With (Application) | Calcium Assay |
| For Use With (Equipment) | Fluorescence Microscope |
| Product Type | Dye |
| Dye Type | Fluorescent Dye-Based |
| Emission | Visible |
| Form | Lyophilized |
| Modification | Un-Modified, Un-Modified |
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Frequently Asked Questions (FAQs)
This is a known drawback of Calcium Crimson, AM (as well as Calcium Orange, AM and Fura Red, AM, which are also in the same emission range). You can try increasing the concentration and washing out of any unlabeled dye from the media, to try to get better signal-to-background. If that fails, we recommend using Rhod-3 AM instead, which has a much better change in signal in that wavelength.
Regardless of the type of live-cell indicator dye (e.g., calcium indicators, pH indicator, metal ion indicators), make sure there is no serum during the loading step, which can prematurely cleave dyes with AM esters and bind dyes non-specifically. Always optimize the dye concentration and staining time with a positive control before you run your test samples, to give the best signal-to-background. Always run a positive control with a buffer containing free ions of known concentration and an ionophore to open pores to those ions (for instance, for calcium indicators like Fluo-4 AM, this would include a buffer with added calcium combined with calcimycin, or for pH indicators, buffers of different pHs combined with nigericin). Reactive oxygen indicators, such as CellROX Green or H2DCFDA would require a cellular reactive oxygen species (ROS) stimulant as a positive control, such as menadione. Finally, make sure your imaging system has a sensitive detector. Plate readers, for instance, have much lower detector efficiency over background, compared to microscopy or flow cytometry.
For Research Use Only. Not for use in diagnostic procedures.
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