Fluo-4
Fluo-4 is a visible light-excitable calcium indicator derived from its predecessor, Fluo-3. Like Fluo-3, Fluo-4 is a non-fluorescent indicator whose intensity increases ~100 fold upon binding Ca2+. Additionally, Fluo-4 is an analog of Fluo-3 exhibiting minor structural modifications attributing to a brighter and more photostable indicator. By replacing two chlorine substituents with fluorines, Fluo-4 displays as wavelength shift of ~12 nm towards the blue spectrum. This establishes a new maximum absorption wavelength at 495 nm making Fluo-4 more efficiently excitable by the 488 nm argon laser line. This attributes to a brighter fluorescence signal at lower dye concentrations making Fluo-4 less phototoxic compared to Fluo-3. Fluo-4 has a slightly higher Ca2+ binding affinity than Fluo-3, with a Kd value of 345 nM. For these reasons, Fluo-4 is better alternative for use with confocal microscopy than Fluo-3.
Fluo-4 AM Esters and Salt Derivatives for Calcium Imaging
Fluo-4 is available as a membrane-permeant acetoxymethyl (AM) ester or as a membrane-impermeant salt. As an AM ester, the increased hydrophobicity of Fluo-4, AM allows it to readily penetrate intact membranes of living cells, and hydrolysis by non-specific intracellular esterases activates Fluo-4's calcium sensitive fluorescence. Cells may also be physically loaded with membrane-impermeant calcium indicators, such as salt derivatives and dextran conjugates. Common cell loading techniques include patch pipette, microinjection or pinocytic loading. Fluo-4 should be stored desiccated and protected from light at ≤ -20°. As received AM esters can typically be stored for at least six months, but beware they are susceptible to hydrolysis, particularly when stored in solution.
Table 1. Fluo-4 green fluorescent calcium indicators for live cell calcium imaging.
Indicator ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Filter Set ▲ ▼ | Kd¹ ▲ ▼ | Φ² ▲ ▼ | FCa/FFree³ ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* | 495 | 528 | FITC | 345 nM | 0.16 | ∼100 fold | 1 mg | 20550 |
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* | 495 | 528 | FITC | 345 nM | 0.16 | ∼100 fold | 10x50 µg | 20551 |
Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* | 495 | 528 | FITC | 345 nM | 0.16 | ∼100 fold | 5x50 µg | 20552 |
Fluo-4, Pentapotassium Salt | 495 | 528 | FITC | 345 nM | 0.16 | ∼100 fold | 1 mg | 20555 |
Fluo-4, Pentapotassium Salt | 495 | 528 | FITC | 345 nM | 0.16 | ∼100 fold | 1 mg | 20556 |
- Kd: Calcium dissociation constant.
- Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
- FCa/FFree: Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.
Screening GPCR and Calcium Channel Targets
ATP Dose Response. 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 (50 µL/well) was added by Flexstation 3 (MDC) to achieve the final indicated concentrations.
Screen Quest™ Fluo-4 Key Features
- Optimized protocols validated for a broad range of microplate readers
- Superior signal intensity (high S/B ratios) and well-to-well uniformity
- Large assay window for assaying challenging cell lines and receptors
Table 2. Screen Quest™ Fluo-4 no-wash calcium assay for screening GPCR and calcium channel targets.
Tips to Improve Results
In past comparative studies, cells loaded with Fluo-4 have displayed favorably large dynamic ranges sensitive at detecting elementary and global Ca2+ fluxes. However, Fluo-4 requires incubation at 37°C resulting in significant indicator loss by the increase activity of anion transporters sensitive to the same temperature. To improve cellular retention reagents such as probenecid can be added to the dye working solution. Probenecid works be inhibiting organic anion transporters located in the cell membranes. Unfortunately, probenecid can be toxic to cells and while it inhibits certain anion transporters it may activate others. For example, the TRPV2 receptors of sensory ganglia have been shown to be activated by probenecid. On the other hand, probenecid seems to have an inhibitory effect on TAS2R receptors, or taste receptors. For many other serum-sensitive or drug-sensitive targets, the effects of probenecid could be largely unknown. This means that by using probenecid, a research risks the introduction of a completely unknown factor into an experimental design.
ATP Dose Response. CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of either 10 µg/mL Calbryte™ 520 AM in HH Buffer with probenecid or 10 µg/mL Fluo-4, AM in HH Buffer with probenecid was added to the wells and incubated for 45 minutes at 37°C. Both dye loading solutions were removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added to achieve the final indicated concentration of 10 µM. Images were acquired on a Keyence microscope in the FITC channel.
High-performance alternative to Fluo-4
Noting this critical problem with existing calcium indicators, Calbryte™ 520 has been specifically designed for high performance without probenecid. Calbryte™ 520 is our upgrade for traditional green fluorescent indicators such as Fluo-3 and Fluo-4. This dye has an excitation maximum at 493 nm, which closely matches the standard 488 nm argon ion laser line. Also, with an emission maximum at 515 nm, this dye is compatible with the FITC filter sets found in most fluorescence instruments. Because Calbryte™ 520 has spectral values nearly identical to that of Fluo-3, no additional instrument setup is required. This allows for a seamless and convenient transition between products.
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Table 3. Calbryte™ 520 green fluorescent calcium indicators for live cell calcium imaging.
Indicator ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Filter Set ▲ ▼ | Kd¹ ▲ ▼ | Φ² ▲ ▼ | FCa/FFree³ ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
Calbryte™ 520 AM | 493 | 515 | FITC | 1.2 µM | 0.75 | ∼300 fold | 2x50 µg | 20650 |
Calbryte™ 520 AM | 493 | 515 | FITC | 1.2 µM | 0.75 | ∼300 fold | 10x50 µg | 20651 |
Calbryte™ 520 AM | 493 | 515 | FITC | 1.2 µM | 0.75 | ∼300 fold | 1 mg | 20653 |
Calbryte™ 520, potassium salt | 493 | 515 | FITC | 1.2 µM | 0.75 | ∼300 fold | 10x50 µg | 20658 |
Calbryte™ 520, potassium salt | 493 | 515 | FITC | 1.2 µM | 0.75 | ∼300 fold | 2x50 µg | 20656 |
- Kd: Calcium dissociation constant.
- Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
- FCa/FFree: Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.
Table 4. Probenecid and Pluronic® F-127 products for improving fluo-3 cell retention and aqueous solubility.
Product ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
ReadiUse™ probenecid *25 mM stabilized aqueous solution* | 10x10 mL | 20062 |
ReadiUse™ probenecid, sodium salt *Water-soluble* | 10x77 mg | 20061 |
Probenecid *Cell culture tested* *CAS 57-66-9* | 10x72 mg | 20060 |
Pluronic® F-127 *20% solution in DMSO* | 10 mL | 20052 |
Pluronic® F-127 *Cell culture tested * | 10 g | 20050 |
Pluronic® F-127 *10% solution in water* | 10 mL | 20053 |