Fluo-4 AM: Comprehensive Guide and Applications
Overview
Fluo-4 AM is a cell-permeable calcium indicator that is metabolized by intracellular esterases, producing a bright green fluorescent signal upon calcium binding (excitation/emission at 494/506 nm). It is ideal for measuring and visualizing intracellular Ca²⁺ in applications like microscopy, flow cytometry, spectrofluorometry, and high-throughput screening.
Spectral Properties
- Excitation Maximum: 494 nm
- Emission Maximum: 506 nm
- Fluorescence Response: Bright green fluorescence upon Ca²⁺ binding.
Key Applications
- Fluorescence Microscopy: Tracks calcium dynamics in live cells.
- Flow Cytometry: Analyzes calcium flux in cell populations.
- High-Throughput Screening: Monitors calcium signaling in drug discovery.
Advantages Over Fluo-3
- Brighter Emission: Allows use at lower concentrations, reducing cell toxicity.
- Higher Cell Permeation: Quickly enters cells for rapid response measurements.
Usage Guidelines
- Preparation: Dissolve in DMSO; typical final concentration is 1-5 μM.
- Incubation: Load cells at 37 °C for 15-60 minutes; re-loading is possible if needed.
Conclusion
Fluo-4 AM’s high sensitivity, bright emission, and ease of use make it an essential tool for studying calcium signaling. Its improved properties over Fluo-3 enhance its utility in cell biology, physiology, and pharmacology research.
Ref:
[1] Gee K.R. et al. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium. 2000. 27(2). 97-106.
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