Mito-laurdan bromide

Guide
(The following is our recommended protocol. This protocol is for guidance only, should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Concentration recommendation: Prepare stock solution at a concentration suitable for dilution to 2.5 μM working concentration.

2. Working Solution Preparation
2.1 Diluent: FluorobriteTM medium.
2.2 Working concentration: 2.5 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Adherent HeLa cells.[1]
3.1.1 Adherent cells: No trypsinization needed before staining; use accutase for detachment only if sorting post-staining.
3.2 Incubation conditions: Incubate cells with 2.5 μM Mito-Laurdan working solution for 20 minutes.
3.3 Washing steps: Wash once with HBSS, then three times with FluorobriteTM containing 10% FBS for six minutes per wash.

4. Controls
4.1 Set up negative control (untreated cells) and vehicle control (cells treated with DMSO diluent only).
4.2 Positive control validates experimental system; blank control excludes reagent fluorescence interference.

5. Detection & Analysis
5.1 Instrument: Confocal microscope.
5.1.1 Excitation wavelength: 352 nm; emission collected at 409-463 nm (ordered channel) and 473-516 nm (disordered channel).
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Calculate Generalized Polarization (GP) using the formula \( GP = \frac{I_{ordered} - I_{disordered}}{I_{ordered} + I_{disordered}} \); decreased GP indicates increased membrane fluidity, while increased GP indicates reduced membrane fluidity.
5.2.2 Fluorescence localization: Targets inner mitochondrial membrane, with high colocalization with MitoTracker Deep Red.
5.2.3 Mito-Laurdan shows blue fluorescence in ordered membrane regions.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.