Lsm image software 3

NK92 cells (1 × 10⁵) were centrifuged with cytospin (Cytofuge) for 6 min at 1000 rpm onto glass cover slides. Before labelling, cells were allowed to recover for 15 min and then fixed with 4% paraformaldehyde for 45 min and permeabilized by 0.1% Triton X-100 in PBS, pH 7.4, for 10 min. Nonspecific staining was blocked with 3% BSA in PBS, pH 7.4, for 1 hour. Lysosomes were labelled by adding LysoTracker to the cells for 2 min before fixation with 4% paraformaldehyde, as recommended by Molecular Probes. All primary antibodies were added after nonspecific blocking. After 2 hours incubation with primary antibody, cells were washed with PBS and treated with fluorophore-labelled secondary antibody for 1 hour. After a final wash with PBS, the ProLong antifade kit (Molecular Probes) was used to mount coverslips on glass slides. Control samples were run in the absence of primary antibodies. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope with 63x Plan-Apochromat Oil DIC objective, N.A. 1.4. Alexa Fluor 488, LysoTracker, or Alexa Fluor 555 was excited with an argon (488 nm) or He/Ne (543 nm) laser, and emission was filtered using narrow-band LP 505–530 nm (green fluorescence) and LP 560 nm (red fluorescence) filters, respectively. Images were analyzed using Carl Zeiss LSM image software 3.0.