Mito-polarity

Operating Instructions (The following is a recommended experimental protocol for guidance only; adjust it according to specific requirements)
1. Stock Solution Preparation
1.1 Solvent: Most dyes are typically dissolved in organic solvents, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.
2. Preparation of Mito-polarity (BOB) Working Solution
2.1 Diluent: Serum-free medium or PBS is usually used. Proteins and esterases in serum may interfere with staining results or cause dye hydrolysis.
2.2 Working Concentration: 2.0 μM.
3. Staining Procedure
3.1 Sample Types^[1]: Adherent cells (MCF-7, HepG2, HeLa, cos-7) and suspension cells (RAW 264.7).
3.2 Incubation Conditions: Incubate cells with 2.0 μM dye solution for 15 min.
4. Detection and Analysis
4.1 Instrument: Confocal fluorescence microscope.
4.1.1 Excitation wavelength Ex = 405 nm; emission wavelengths Em = 435-535 nm (green channel), Em = 575-675 nm (red channel).
4.2 Result Analysis:
4.2.1 Changes in fluorescence intensity: A decrease in solution polarity leads to an increase in green fluorescence intensity (467 nm/468 nm), while red fluorescence intensity (642 nm/645 nm) shows a slight change; the fluorescence intensity ratio (I₄₆₇/I₆₄₅) decreases with reduced polarity.
4.2.2 Fluorescence localization: Fluorescence localizes specifically to mitochondria, which is verified by colocalization with MitoTracker Green FM (HY-135056) (Pearson correlation coefficient R = 0.96).
4.2.3 Color changes: Green fluorescence represents higher polarity, while red fluorescence signal remains stable across different polarity ranges; ratio images indicate polarity via color gradients (green for high polarity, magenta for low polarity).
4.2.4 Expected results of cell experiments: The mitochondrial polarity of normal cells (cos-7, RAW 264.7) is higher than that of cancer cells (HeLa, HepG2, MCF-7); the mitochondrial polarity of dying/apoptotic cells is lower than that of normal cells.

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