FM4-64

The lipophilic FM4-64 dye [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridiumdibroide] has basically a polyethylene structure. This water-soluble dye is non-toxic to live tissues according to the manufacturer (Thermofisher). Cell viability was also confirmed using trypan blue. In pure water the dye was solved to a stock solution (1 μg/μl). The staining was optimized over various experiments. An incubation time of 10–15 min was selected together with ambient temperature in protoplast buffer and a 0.5% (v/v) final concentration for the staining of olive protoplasts. The fluorophore was excited at a wavelength of 543 nm using an FM4-64 emission spectrum in the 580–650 nm range (emission maximum: 640 nm).