Fluo 3-AM | Dojindo

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Fluo 3-AM

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Item #	Description/Size	Availability	Qty Break	Price	Quantity
F023-10	1 mg	5-10 business days	1	$318.00

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Description
Application

Chemical Name: 1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, pentaacetoxymethyl ester
CAS: 121714-22-5

Appearance: Red powder or solid
Purity: ≥85.0 %(HPLC)
MW: 1129.85, C₅₁H₅₀Cl₂N₂O₂₃

Storage Condition: -20ºC, protect from light and moisture
Shipping Condition: ambient temperature

Product Description
Fluo 3 is a long wavelength calcium probe that is practically nonfluorescent in its free ligand form, but its fluorescence increases 60-80 times when it forms complexes with calcium. Thus, it has been widely used with confocal laser fluorescent microscopy because the microscope has an argon laser. The long wavelength of the fluorescent signal is also convenient for minimizing photodamage to sample cells. Fluo 3 is also useful for caged calcium and others that are cleaved by the photoirradiation in the UV region. Fluo 3-AM is an acetoxymethyl ester derivative of Fluo 3 that can be easily loaded into cells by incubation.

Hydrolysis of AM ester

General Protocol (for Human T cells)*
Reagents:

2 mM Fluo 3-AM/DMSO (1 mg Fluo 3-AM in 442 μl DMSO)
Pluronic F127
Hanks Ebalanced salt solution (HBSS)
HEPES buffer saline (10 mM HEPES, 1 mM Na₂HPO₄, 137 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 5 mM glucose, 0.1% BSA, pH 7.4)

Protocol:

Add 16.5 mg Pluronic F127 to Fluo 3-AM/DMSO solution. Pluronic F127 prevents aggregation of Fluo 3-AM in HBSS and helps uptake with cells.
Dilute the Fluo 3-AM solution with HBSS to prepare 4 μM Fluo 3-AM working solution.
Add the Fluo 3-AM working solution to the cells and incubate at 37^oC for 20 min.
Add HBSS containing 1% fetal Calf serum equivalent to 5 times the volume of Fluo 3 -AM working solution (step 3).
Wash the cells 3 times with HEPES buffer saline. Then resuspend the cells to prepare 1x10⁵ cells per ml solution using HEPES buffer saline.
Incubate at 37^oC for 10 min. Then use the cells for fluorescent calcium ion detection.
Monitor the fluorescence at 528 nm (excitation: 490-500 nm).

*Cell staining conditions depend on cell types, so it is necessary to optimize the conditions for each experiment.

References
1. A. Minta, et al., Fluorescent Indicators for Cytosolic Calcium Based on Rhodamine and Fluorescein Chromophores. J Biol Chem. 1989;264:8171-8178.
2. J. P. Kao, et al., Photochemically Generated Cytosolic Calcium Pulses and Their Detection by Fluo-3. J Biol Chem. 1989;264:8179-8184.
3. M. Eberhard, et al., Kinetics of Calcium Binding to Fluo-3 by Stopped-Flow Fluorescence. Biochem Biophys Res Commun. 1989;163:309-314.
4. A. Hernandez-Cruz, et al., Subcellular Calcium Transients Visualized by Confocal Microscopy in a Voltage-clamped Vertebrate Neuron. Science. 1990;247:858-862.
5. A. H. Cornell-Bell, et al., Glutamate Induces Calcium Waves in Cultured Astrocytes: Long-Range Glial Signaling. Science. 1990;247:470-473.
6. D. A. Williams, Quantitative Intracellular Calcium Imaging with Laser-scanning Confocal Microscopy. Cell Calcium. 1990;11:589-597.
7. D. A. Williams, et al., Confocal Imaging of Ionised Calcium in Living Plant Cells. Cell Calcium. 1990;11:291-297.
8. P. A. Vandenberghe, et al., Flow Cytometric Measurement of Cytoplasmic Free Calcium in Human Peripheral Blood T Lymphocytes with Fluo-3, A New Fluorescent Calcium Indicator. J Immunol Methods. 1990;127:197-205.
9. M. Iino, et al., Visualization of Neural Control of Intracellular Ca2+ Concentration in Single Vascular Smooth Muscle Cells in situ. EMBO J. 1994;13:5026-5031.
10. M. E. Dailey, et al., Spontaneous Ca2+ Transients in Developing Hippocampal Pyramidal Cells. J Neurobiol. 1994;25:243-251.
11. M. Burnier, et al., Confocal Microscopy to Analyze Cytosolic and Nuclear Calcium in Cultured Vascular Cells. Am J Physiol. 1994;266:C1118-C1127.
12. E. Donnadieu, et al., Ca2+ Signaling in Endothelial Cells Stimulated by Bradykinin: Ca2+ Measurement in the Mitochondria and the Cytosol by Confocal Microscopy. Cell Calcium. 1996;20:53-61.
13. M. Ikeda, et al., Separate Analysis of Nuclear and Cytosolic Ca²⁺ Concentrations in Human Umbilical Vein Endothelial Cells. J Cell Biochem. 1996;63:23-36.
14. J. E. Merritt, et al., Use of fluo-3 to Measure Cytosolic Ca²⁺ in Platelets and Neutrophils Loading Cells with the Dye, Calibration of Traces, Measurements in the Presence of Plasma, and Buffering of Cytosolic Ca2+. Biochem J. 1990;269:513-519.