BV-1

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

1. Stock Solution Preparation
1.1 Solvent: Ethanol or DMSO.
1.2 Recommended concentration: 1 × 10^-3 M (1 mM).

2. Working Solution Preparation
2.1 Diluent: Cell culture medium.
2.2 Working concentration: 5 μM.
2.3 Notes: Adjust the working solution concentration as required; prepare and use immediately.

3. Staining Procedures
3.1 Applicable to adherent cells:
3.1.1 Sample type: Adherent cells (A549); seed the cells in a culture plate before staining; trypsinization is not required for staining.
3.1.2 Incubation conditions: Incubate at 37°C for 30 min; for A549 cells, incubate in a humidified environment containing 5% CO₂; no need for light protection is mentioned in literature.
3.1.3 Washing step: Wash 3 times with phosphate-buffered saline (PBS) after incubation.

4. Control Setup
4.1 Unstained control: Used to adjust instrument voltage and eliminate autofluorescence of cells.
4.2 Blank control: Only add BV-1 without drug treatment, used to exclude fluorescence changes caused by the dye itself.

5. Detection and Analysis
5.1 Instrument type: Laser confocal microscope, confocal fluorescence microscope.
5.2 Excitation/emission wavelength: Ex = 488 nm; Em = 525-650 nm.
5.3 Result analysis:
5.3.1 Changes in fluorescence intensity: The higher the environmental viscosity, the stronger the fluorescence intensity; a time-dependent fluorescence enhancement can be observed during the increase of cellular viscosity.
5.3.2 Fluorescence localization: The fluorescence of BV-1 localizes to mitochondria, showing red fluorescence; the fluorescence is weak at low viscosity, while it increases significantly with an emission redshift at high viscosity.