RhoNox-1 is a divalent iron ion (Fe²⁺) fluorescent probe specific and available for use in living cells, with a maximum absorption wavelength of 540 nm and a maximum emission wavelength of 575 nm, producing an irreversible orange-red fluorescent product. RhoNox-1 has the advantage of cell membrane permeability and is usually localized to the c.

Instructions
I. Solution preparation
1. Preparation of mother solution: Use DMSO to prepare RhoNox-1 and obtain 1 mM stock solution.
Note: Please adjust the concentration of the mother solution according to your experimental needs. The mother solution should be stored in aliquots at -80℃ or -20℃ to avoid repeated freezing and thawing.
2. Preparation of working solution: Dilute the stock solution with preheated serum-free cell culture medium or PBS to prepare 1-10 μM RhoNox-1 working solution.
Note: Please adjust the concentration of RhoNox-1 working solution according to actual conditions and prepare it before use.
II. Cell staining
1. Adherent cells
1) Culture adherent cells on sterile coverslips.
2) Remove the coverslips from the culture medium and remove excess culture medium.
3) Add 100μL of dye working solution, shake gently to completely cover the cells, and incubate at room temperature for 5-30 minutes.
4) Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometer.
2. Suspended cells
1) Suspended cells: Collect cells by centrifugation, add PBS and wash twice, 5 minutes each time. Cell density is 1×106/mL
2) Add 1 mL of working solution and incubate at room temperature for 5-30 minutes.
3) Centrifuge at 400 g for 3-4 minutes and discard the supernatant.
4) Add PBS and wash cells twice, 5 minutes each time.
5) Resuspend cells with 1 mL of serum-free culture medium or PBS and observe using a fluorescence microscope or flow cytometer.