Gol-SiRhoNox

Operating Instructions (The following is a recommended experimental protocol for guidance only; please adjust it according to your specific requirements)

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

2. Working Solution Preparation
2.1 Diluent: HBSS.
2.2 Working concentration: 5 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample Type Description
3.1.1 Adherent cells (HepG2 cells, SH-SY5Y neuroblastoma cells)^[1].
3.2 Incubation Conditions
3.2.1 For adherent cells: Incubate cells with 5 μM Gol-SiRhoNox at 37°C for 30 min.
3.3 Washing Steps
3.3.1 For adherent cells: Before adding the probe, carefully wash the cells 3 times with an appropriate buffer to remove extracellular Fe (II).

4. Control Setup
4.1 Positive control: Before probe incubation, incubate cells in FBS-free Modified Essential Medium (MEM) with 100 μM Fe (II) (added as Fe (NH₄)₂ (SO₄)₂·6H₂O) at 37°C for 30 min.
4.2 Negative control: Incubate cells with Gol-SiRhoNox in a system containing 1 mM 2,2'-Bipyridine (HY-D0020).
4.3 Blank control: Do not perform Fe (II) supplementation treatment on cells before probe incubation.

5. Detection and Analysis
5.1 Instrument type: Fluorescence microscope.
5.2 Excitation/emission wavelength: 630 nm/665 nm (Cy5 filter set).
5.3 Result Analysis
5.3.1 Fluorescence localization: Fe (II) mainly distributes in the Golgi apparatus in normal cells, while its localization shifts to lysosomes in systems with protein sorting defects.
5.3.2 Changes in fluorescence intensity: Fluorescence is generated by the interaction with labile Fe (II) in the Golgi apparatus; cells pretreated with Fe (II) show increased fluorescence intensity; cells treated with Fe (II) chelators show decreased fluorescence intensity; the Golgi fluorescence intensity of VPS35-knockdown cells is lower than that of wild-type cells, and it can be restored after treatment with R55.
5.3.3 Color change: Magenta fluorescence is observable under the Cy5 filter set.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.