Ac-MtFluNox

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: 1 mM.

2. Working Solution Preparation
2.1 Diluent: HBSS; MEM without FBS.
2.2 Working concentration: 1 μM, 2 μM, 5 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type descriptions
3.1.1 For adherent cultured cells^[1][2]: Includes HepG2 cells, HEK293 cells, HT1080 cells; no trypsinization required prior to staining.
3.2 Incubation conditions
3.2.1 For adherent cultured cells[1]: Incubate with 1-5 μM Ac-MtFluNox in HBSS or MEM without FBS at 37°C for 30 min-3 h; co-staining with 0.5 μM MitoTracker® Deep Red FM is optional during incubation.
3.3 Washing steps
3.3.1 For adherent cultured cells[1]: Wash once with MEM without FBS before incubation; wash once with HBSS after incubation.

4. Controls
4.1 Set up negative controls using equal volumes of vehicles instead of Fe(II), succinylacetone, or NOC-5.
4.2 Set up competitive inhibition controls with Fe(II) chelator 2,2′-bipyridyl or iron scavenger deferroxamine.

5. Detection & Analysis
5.1 Instrument type: Confocal fluorescence microscope; fluorescence microscope.
5.2 Ex/Em wavelengths: Excitation 465-500 nm, emission 516-556 nm (FITC filter set).
5.3 Result analysis
5.3.1 Fluorescence intensity changes: Turn-on fluorescence enhancement upon reaction with mitochondrial labile Fe(II); intensity is reduced in the presence of Fe(II) chelators.
5.3.2 Fluorescence localization: Fluorescence colocalizes with mitochondria (confirmed by co-staining with MitoTracker® Deep Red FM).
5.3.3 Color changes: Green fluorescence; fluorescence emission specific to labile Fe(II) detection via N-oxide deoxygenation mechanism.