BODIPY FL-DHPE

Operating Instructions (The following is a recommended experimental protocol for guidance only, and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: Chloroform.
1.2 Concentration Recommendation: The mixing molar ratio of BODIPY FL-DHPE to DOPE/DOTAP is 1/1/0.01-0.1 mol/mol.
1.3 Notes: Avoid repeated freeze-thaw cycles.

2. Working Solution Preparation
2.1 Diluent: The liposome stock solution is diluted with PBS (pH 7.4) (1/50 dilution) for cell incubation; it can also be diluted with cell culture medium (1/100 dilution).
2.2 Working Concentration: The total lipid concentration is 0.02 mg/mL (for the lipid mixture stock solution).
2.3 Notes: Adjust the working solution concentration as needed; prepare it immediately before use.

3. Staining Procedure
3.1 Sample Type Description: CHO cells, HEK293 cells, myofibroblasts, cortical neurons, macrophages, smooth muscle cells, keratinocytes, HeLa cells, MEF, etc.
3.2 Incubation Conditions: Aspirate the culture medium, add the diluted liposome solution (pH 7.4), and incubate in a 37 °C, 5% CO₂ incubator.
3.3 Washing Steps: After incubation, directly replace with fresh medium for imaging; before flow cytometry detection, digest cells with trypsin-EDTA for 2-5 min, wash with PBS (pH 7.4), and centrifuge at 200 ×g for 5 min.

4. Control Setup
4.1 Unstained Control: Use untreated cells to adjust the photomultiplier tube voltage, so that the signal intensity of green and red channels is lower than 10 counts, thereby eliminating cell autofluorescence.
4.2 Endocytosis Control: Prepare non-fusogenic (endocytic) liposomes by replacing DOPE with DMPC as a control for the endocytic pathway; cells treated with endocytic liposomes show punctate green signals, with a high dimer/monomer ratio.

5. Detection and Analysis
5.1 Instrument Types: Laser confocal microscope and flow cytometer.
5.2 Excitation/Emission Wavelengths
5.2.1 Confocal Microscope: Excitation at 488 nm, BP 495-550 nm (monomer / green), LP 619 nm (dimer / red).
5.2.2 Flow Cytometer: Excitation at 488 nm, 525/30 nm (monomer / green), 690/50 nm (dimer / red).
5.2.3 Fluorescence Spectrophotometer: Excitation at 488 nm, emission at 500-700 nm.
5.3 Result Analysis
5.3.1 Changes in Fluorescence Intensity: After membrane fusion, BODIPY FL-DHPE dilutes in the plasma membrane, leading to enhanced fluorescence at the monomer peak (515 nm) and weakened fluorescence at the dimer peak (620 nm); after endocytosis, the dye remains in endosomes with high local concentration, so the dimer peak remains unchanged. The fluorescence intensity ratio of green/red channels serves as a reliable indicator of cellular uptake pathways (fusion vs endocytosis).
5.3.2 Fluorescence Localization: During membrane fusion, fluorescence distributes uniformly across the entire plasma membrane, clearly outlining the cell; during endocytosis, fluorescence shows punctate distribution inside the cell, corresponding to endosomal localization.
5.3.3 Color Changes: At low concentrations (≤5 mol%), green monomer fluorescence dominates; at high concentrations (≥5 mol%), red dimer fluorescence appears. After fusion, the red dimer signal weakens while the green monomer signal enhances; during endocytosis, the ratio of red to green signals remains unchanged.