Visualizing Autolysosomes in Tobacco Cells

TBY-2 suspension tobacco cells (200 µL) were centrifuged at 4000× g for 5 min and the pellet was resuspended in 200 µL of 100 mM potassium phosphate buffer (PK) pH 6.5 containing 1 µM of Lysotracker Deep Red (Invitrogen, Thermo Fisher, Madrid, Spain) as previously described [36 (link)]. The mixture was incubated in the dark and at room temperature for 6 min. Subsequently, the preparation was centrifuged at 1000× g for 1 min, the supernatant was discarded, and the pellet was washed three times with PK buffer, pH 6.5 at 1000× g for one minute, and the final precipitate was resuspended in 150 µL of the same buffer. Fifty microliters were added to the slide and the structures were visualized in a Leica DM6 motorized fluorescence microscope equipped with Nomarski optic and a digital Hamamatsu C11440 camera. For the detection of autolysosomes, the preparations were observed using a band pass (BP) 545–580, DM 600, and a BA filter of 610. At least ten fields (approximately 20 cells per field) were evaluated in each of the three independent experiments carried out. Image analysis was performed using ImageJ software (Softonic, NIH, Bethesda, MD, USA).