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Classic Calcium Indicators
Classic calcium indicators remain popular tools for measuring intracellular calcium dynamics across a wide range of experimental platforms. AAT Bioquest offers a comprehensive selection of these indicators, including Fura-2, Indo-1, Fluo-4, Rhod-2, and their variants, available in multiple forms (AM esters, salts) and purity grades (standard and UltraPure) to meet diverse experimental requirements. These probes support applications ranging from ratiometric quantitative measurements to single-wavelength high-throughput screening.
Ratiometric Calcium Indicators
Examples of ratiometric calcium indicators include Fura-2 and Indo-1. These probes enable quantitative measurement of intracellular calcium concentrations while substantially reducing artifacts arising from uneven dye loading, cell thickness, and photobleaching. Fura-2 exhibits an excitation wavelength shift upon calcium binding (from 340 nm to 380 nm), while Indo-1 shows an emission wavelength shift (from 405 nm to 485 nm), making them ideal for different instrument configurations.
Key Features
- Quantitative measurements: Ratiometric detection eliminates artifacts from uneven dye loading and photobleaching
- UltraPure grades available: Higher purity for demanding applications requiring optimal signal-to-noise
- Fura-FF variant: Lower calcium affinity (Kd ~5.5 µM vs ~145 nM of Fura-2) for measuring higher calcium concentrations
- Multiple salt forms: Pentapotassium and pentasodium salts for microinjection and in vitro calibration
- Indo-1 advantage: Emission-ratiometric design ideal for flow cytometry with single UV laser excitation
Single-Wavelength Calcium Indicators
Examples of single-wavelength calcium indicators include Fluo-4 and Rhod-2. These indicators are compatible with standard visible-light excitation sources (e.g., 488 nm argon laser for Fluo-4). Upon calcium binding, single-wavelength calcium indicators exhibit large fluorescence increases, making them excellent choices for detecting calcium transients, though absolute quantification is limited by their single-wavelength detection mode. Fluo-4 provides bright green fluorescence ideal for confocal microscopy and high-throughput screening, while Rhod-2's red emission enables multiplexing with green fluorescent probes and preferential localization in mitochondria.
Key Features
- 488 nm compatible: Fluo-4 works with argon lasers, FITC filter sets, and standard plate readers
- UltraPure grade Fluo-4: Exceptional signal-to-noise ratio for demanding imaging applications
- Rhod-2 for mitochondria: Positive charge promotes preferential accumulation in mitochondria for organelle-specific Ca²⁺ measurements
- Dextran conjugates: Rhod-2 dextrans provide stable retention in neurons for presynaptic calcium imaging
- Large dynamic range: Both indicators show >100-fold fluorescence increase upon calcium binding
Low-Affinity Calcium Indicators
Mag-Fura-2 and Mag-Fluo-4 are low-affinity calcium indicators originally developed for intracellular magnesium measurements. With calcium Kd values in the micromolar range (~22 µM for Mag-Fluo-4), these indicators are well suited for measuring high calcium concentrations found in organelles such as the endoplasmic reticulum and sarcoplasmic reticulum, or in systems where cytosolic calcium reaches high levels during stimulation.
Key Features
- High [Ca²⁺] detection: Ideal for measuring ER/SR calcium stores where concentrations reach high µM range
- Dual ion capability: Can also serve as intracellular magnesium indicators (Kd ~4.7 mM for Mg²⁺)
- Mag-Fura-2: Ratiometric UV-excitable indicator matching Fura-2 spectral properties
- Mag-Fluo-4: Single-wavelength, visible-light excitable (488 nm) for standard confocal and plate reader compatibility
Screen Quest™ No Wash Calcium Assay Kits
Screen Quest™ No Wash Calcium Assay Kits provide optimized, ready-to-use formulations for high-throughput GPCR screening and calcium flux assays. These kits incorporate classic calcium indicators (Fluo-4 or Fura-2) with proprietary masking dyes that quench extracellular fluorescence, allowing no-wash detection of intracellular calcium signals.
Key Features
- No-wash protocol: Significantly reduces assay time and improves throughput for HTS applications
- Background suppression: Proprietary masking dye quenches extracellular indicator fluorescence
- GPCR screening optimized: Ideal for calcium-mobilizing receptor studies in drug discovery
- Flexible format: Available with either Fluo-4 (single-wavelength) or Fura-2 (ratiometric) indicators
- Complete kit: Includes all reagents needed for cell-based calcium flux assays
Fig. 2
ATP Dose Response was measured in CHO-K1 cells with Screen Quest™ Fluo-4 No Wash Calcium Assay Kit. CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo-4 No Wash calcium assay kit for 1 hour at room temperature. ATP (50µL/well) was added by Flexstation 3 (MDC) to achieve the final indicated concentrations.
This document (01.0238.251203r1) was last updated on Fri Feb 13 2026. All trademarks and registered trademarks mentioned herein are the property of their respective owners.