BODIPY-DOH

Operation Guide (The following is a recommended experimental protocol for guidance only and should be adjusted according to your specific needs)
1. Stock Solution Preparation
1.1 Solvent: anhydrous DMSO^[1]; tetrahydrofuran^[2].
1.2 Recommended concentration: Generally, a high-concentration stock solution of 1-10 mM is recommended.
2. Working Solution Preparation
2.1 Diluent: Serum-free medium or PBS is typically used. Proteins and esterases in serum may interfere with the staining effect.
2.2 Working concentration: 50-400 nM^[2].
2.3 Notes: The working solution concentration can be adjusted as needed; it should be prepared fresh and used immediately.
3. Staining Procedure
3.1 Sample Type Description
3.1.1 Adherent cells^[2]: No trypsin digestion step is required for imaging; cells need to be collected for flow cytometry. For primary hippocampal neurons, if fixation and imaging are required after incubation, cells can be fixed with 4% PFA.
3.2 Incubation Conditions
3.2.1 Adherent cells^[2]: Incubate undamaged or glutamate-damaged hippocampal neurons with BODIPY-DOH working solution
(50 nM, 100 nM, 200 nM, or 400 nM) at 37°C, 5% CO₂ in the dark.
3.3 Washing Steps
3.3.1 Adherent primary hippocampal neurons^[2]: After incubation with the ROS detection reagent, wash the cells with PBS.
4. Control Setup
4.1 Unstained control^[1]: Used to adjust instrument voltage and eliminate cell autofluorescence.
4.2 Positive control (elevated O₂^•- )^[1]: Treat cells with Phorbol 12-myristate 13-acetate (PMA) (HY-18739) to stimulate intracellular O₂^•- production.
Expected result: Significant quenching of cellular fluorescence and decreased fluorescence intensity.
4.3 Reduction control (elevated GSH)^[1]: Treat cells with α-Lipoic Acid (LPA) (HY-N0492) to increase intracellular L-Glutathione reduced (GSH) (HY-D0187) levels.
Expected result: Fluorescence recovery to initial levels.
5. Detection and Analysis
5.1 Instrument types^[2]: Confocal fluorescence microscope; flow cytometer.
5.2 Excitation/Emission wavelengths
5.2.1 Fluorescence detection^[2]: Excitation wavelength Ex = 633 nm.
5.3 Result analysis
5.3.1 Fluorescence intensity analysis^[2]: After BODIPY-DOH staining, cells exhibit red fluorescence under normal conditions, and the fluorescence co-localizes with neuronal tubulin in the cytoplasm and processes; when O₂^•- levels increase and oxidative stress intensifies, the red fluorescence intensity decreases significantly; when GSH levels increase, the fluorescence recovers. In damaged neurons, the fluorescence intensity of BODIPY-DOH decreases with increasing oxidative stress levels (higher glutamate concentrations), and the red fluorescence intensity of undamaged neurons is significantly higher than that of the damaged group. The dynamic changes in red fluorescence intensity reflect changes in the intracellular redox state.