■ Preparation of reagent and cell imaging

1. Before opening the vial, spin down the solid to the bottom by a microcentrifuge or a desktop centrifuge.

2. 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 DMSO solution.

3. Dilute the FeRhoNox™-1 solution with HBSS or with other neutral buffer solution to prepare 5 μM solution. (The concentration of the probe should be changed depending on the measurement conditions and samples.)

4. Remove culture medium from the cells cultured in a glass-bottom dish, rinse the cells with HBSS twice, then add the 5 μM FeRhoNox™-1 solution.

5. Incubate cells for 37oC, 5% CO2 for 60 minutes. Incubation time should be varied depending on the conditions.

6. Rinse the cells with HBSS three times and observe cells by microscopy.

※ You can detect the increase of labile Fe2+ ions if you added Fe2+ in the medium. For this purpose, dissolve Fe(NH4)2(SO4)2 (FAS) to prepare a 100 mM solution just before use, then dilute the FAS solution 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 intracellular Fe2+.

■ Fluorescence observation

Use green excitation filter set for Cy3 or tetramethyl rhodamine (TMR). For laser excitation, 532 nm or 543 nm laser is appropriate. Detect fluorescence around 570 nm.

Table 2. Related Products