Mito-RhFe

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

1. Stock Solution Preparation
1.1 Solvent: DMSO.

2. Working Solution Preparation
2.1 Diluent: PBS buffer.
2.2 Working concentration: 10 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type:[1]
3.1.1 Adherent cells (MCF-7, HeLa): No trypsinization required for imaging.
3.1.2 Suspension cells (MEL, K562): Collect cells, rinse with PBS 3 times before staining.
3.2 Incubation conditions:
3.2.1 For adherent cells: Incubate with 10 μM Mito-RhFe at ambient temperature for 30 min.
3.2.2 For suspension cells: Incubate with 10 μM Mito-RhFe on ice for 30 min.
3.3 Washing steps:
3.3.1 For suspension cells: Rinse with PBS 3 times after staining.

4. Controls
4.1 For K562 cell assay: Set up control group of cells cultured without DMSO.

5. Detection & Analysis
5.1 Instrument:
5.1.1 Confocal laser scanning fluorescence microscope: Ex = 543 nm, Em = 570–620 nm.
5.1.2 Flow cytometer: Use PE channel.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Fluorescence intensity increases with elevated mitochondrial labile Fe³⁺ levels; intensity decreases when labile Fe³⁺ is scavenged by TPEN.
5.2.2 Fluorescence localization: Fluorescence is localized to mitochondria, confirmed by colocalization with MitoTracker Deep Red 633.
5.2.3 Color changes: Solution changes from colorless to magenta upon Fe³⁺ binding in vitro.