FM4-64

The lipophilic FM4-64 dye (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridiumdibromide) is based on a polyethylene structure. FM4-64 is employed extensively for studying vesicle organelle organization, trafficking, and endocytosis in eukaryotic cells (Bolte et al., 2004). According to the manufacturer (Thermofisher), the dye, water soluble, is non-toxic for living cells; cell viability was corroborated by Trypan blue application, after solving the dye in purified water to a stock solution of 1 μg/μl. After several experiments to optimize staining, a final concentration of 0.5% (v/v) and an incubation period of 10 to 15 min at room temperature in protoplast buffer were selected to stain the olive protoplasts. A wavelength of 543 nm was employed to excite the fluorophore; the emission spectrum of FM4-64 was in the range of 580–650 nm (emission maximum 640 nm).