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.