Mito-VR2

Operating Instructions
(The following is a recommended experimental protocol for guidance only, and needs to be adjusted according to your specific requirements).

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Recommended concentration: 1 mM

2. Working Solution Preparation
2.1 Diluent: PBS
2.2 Recommended concentration: 1.0 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type: Adherent cells (HeLa cells)^[1]
3.2 Incubation conditions: Incubate with 1.0 μM Mito-VR2 for 30 min.
3.3 Washing step: Wash once with PBS after incubation.

4. Control Setup
4.1 The following controls are required for HeLa cell imaging:
4.1.1 Vehicle-only control: Used to confirm the stability of the probe under basic cellular conditions and obtain the background fluorescence signal of cells.
4.1.2 Normally cultured cell control group: After staining, cells are continuously cultured in complete medium without starvation treatment, which is used for comparison with the starvation-induced mitophagy group.
4.1.3 Probe single-staining control: Stain with Mito‑VR2 only, without adding organelle localization reference dyes, which is used to rule out channel crosstalk.
4.1.4 Localization co-staining control: Co-stain with Mito‑VR2 + MitoTracker Green, which is used to verify the mitochondrial targeting localization of the probe.

5. Detection and Analysis
5.1 Instrument: Confocal fluorescence microscope or flow cytometer.
5.1.1 Excitation wavelength: 488 nm; Emission wavelength: ~660 nm.
5.2 Result analysis:
5.2.2 Fluorescence intensity increases with the elevation of microenvironmental viscosity.
5.2.2 Fluorescence localizes specifically to mitochondria.
5.2.3 Red fluorescence is emitted.

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