Single dye staining of protoplasts. Single dye staining of protoplasts, using FM4-64 (A; C), LRB-PE (E), DiIC12 (G), DiIC18 (I), and DiD (K); all dyes appeared at the plasma membrane. Upon FM4-64 treatment first fluorescent vesicles appeared in the cytoplasm within 20 minutes; endocytosis events were strongly enhanced with ongoing incubation time (C; 30 minutes post FM4-64 incubation). All staining protocols were designed with respect to cell viability; macroscopically the protoplasts were not affected and maintained their round shape (B; D; F; H; J; L). Viability was successfully tested using trypan blue.