RhoNox-1, a fluorescent probe, specifically detects divalent iron ions (Fe 2+) by generating an irreversible orange (red) fluorescent product with an excitation and emission maxima at 540 and 575 nm respectively (Ex/Em: 540/575 nm). Upon reacting with Fe 2+, RhoNox-1 efficiently permeates cells, making it ideal for monitoring Fe 2+ in living cells. The probe predominantly localizes in the Golgi apparatus [1].

RhoNox-1 Working Solution Preparation: 1.1 To prepare a storage solution, dissolve 50 μg of RhoNox-1 in 110 μL anhydrous DMSO to achieve a 1 mM stock solution. 1.2 For the working solution, dilute the stock solution with pre-warmed serum-free cell culture medium or PBS to a final concentration of 1-10 μM RhoNox-1. Note: Adjust the concentration as necessary and prepare freshly before use.

Cell Staining (Suspension Cells): 2.1 Collect suspension cells via centrifugation and wash twice with PBS, each for 5 minutes, maintaining a cell density of 1×10^6/mL. 2.2 Incubate with 1 mL of the working solution at room temperature for 5-30 minutes. 2.3 Centrifuge at 400 g for 3-4 minutes and discard the supernatant. 2.4 Wash the cells twice with PBS, each wash lasting 5 minutes. 2.5 Resuspend the cells in 1 mL serum-free medium or PBS and analyze using a fluorescence microscope or flow cytometer.

Cell Staining (Adherent Cells): 3.1 Culture adherent cells on a sterile coverslip. 3.2 Remove the coverslip from the culture medium and aspirate the excess. 3.3 Add 100 μL dye working solution, gently agitate to cover cells fully, and incubate at room temperature for 5-30 minutes. 3.4 Aspirate the dye solution, wash 2-3 times with medium for 5 minutes each, and observe with a fluorescence microscope or flow cytometer.