FeRhoNox™-1 (also called as RhoNox-1) is an activatable fluorescent probe that specifically detects labile Fe2+ ions via orange (red) fluorescence. It irreversibly fluoresces upon reaction with Fe2+ ions, but does not react with other ions of physiological concentrations. FeRhoNox™-1 tends to localize in Golgi within a cell.
Storage
This product is shipped as a dried solid in nitrogen gas-filled vials. Upon receipt, store the product desiccated andprotectedfromlightat≤−20°C.Storingasasolutionisnotrecommendedbecausethereagentdegrades in solution and fluorescence background signal may increase. Please use up thesolution.
An example of live cellimaging
Materials required but notprovided
・Dimethyl sulfoxide (DMSO). We strongly recommend using high-quality grade named as infinity-pure or ultra-pure. DMSO bottle should be opened just before use. Storing DMSO in a deep freezer as a small aliquot is also recommended. Degraded DMSO may increase the background signal of FeRhoNox™-1. ・Observation buffer (1×PBS pH 7.4, Hank’s Balanced Salt Solution (HBSS), etc.).
Preparation of reagent and cellimaging
Before opening the vial, spin down the solid to the bottom by a microcentrifuge or a desktopcentrifuge.
Allow the vial to attain room temperature and add 109 μl of high-purity DMSO to one vial to make 1 mM solution. Use neutral buffer solution for the dilution of the DMSOsolution.
DilutetheFeRhoNox™-1solutionwithHBSSorwithotherneutralbuffersolutiontoprepare5μMsolution. (The concentration of the probe should be changed depending on the measurement conditions and samples.)
Remove culture medium from the cells cultured in a glass-bottom dish, rinse the cells with HBSS twice, then add the 5 μM FeRhoNox™-1solution.
Incubate cells for 37oC, 5% CO2 for 60 minutes. Incubation time should be varied depending on the conditions.
Rinse the cells with HBSS three times and observe cells bymicroscopy.
※ You can detect the increase of labile Fe2+ ions if you added Fe2+ in the medium. For this purpose, dissolveFe(NH4)2(SO4)2(FAS)topreparea100mMsolutionjustbeforeuse,thendilutetheFASsolution with cell culture medium to prepare 100 μM FAS solution. After cells were cultured in the FAS solution for 30 minutes, wash the cells to remove extracellular FAS and add FeRhoNox™-1 solution to detect intracellularFe2+.
Fluorescenceobservation
UsegreenexcitationfiltersetforCy3ortetramethylrhodamine(TMR).Forlaserexcitation,532nmor543nm laser is appropriate. Detect fluorescence around 570nm.
Table 2. Related Products
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