HMRhoNox-M (also known as LysoRhoNox) is a Fe2+-selective fluorescent probe. In the absence of Fe2+, HMRhoNox-M exists in the non-fluorescent form showing only negligible fluorescence in an aqueous buffer and at physiological pH. The addition of Fe2+ induces a 60-fold increase of the fluorescence at 575 nm by the transition of the probe to a fluorescent form. HMRhoNox-M responds to Fe2+ in a dose-dependent manner. It is the cell-permeant probe that is mainly localized in lysosomes.

Here is a brief description of the protocol we used to detect Fe(II) in live cells with HMRhoNox-M:

1. To prepare the 1 mM stock solution of dye, 50 μg of HMRhoNox-M (MW 388.47) was dissolved in 128 μL of DMSO.

2. A549 cells (0.4 million cells per mL) were seeded in DMEM medium with 10% fetal calf serum for 24 h.

3. Cells were incubated for 30 min in DMEM medium (without serum) :

- without any additives;

- with 100 μM Fe(II);

- with 5 μM transferrin (iron-loaded).

4. After three washing with Hank's solution, the cells were incubated for 30 min with 1 μM HMRhoNox-M solution in a CO2 incubator.

5. DAPI (1 μg/mL) was added to cells 10 min before the end of incubation.

6. Cells were washed with Hank's solution and analyzed using a fluorescent microscope at 100x magnification.

A549 cells stained with Fe(II)-selective fluorescent probe HMRhoNox-M (3317-x) and DAPI.

Adding Fe(II) induces a 60-fold increase in the HMRhoNox-M fluorescence.