Lysotracker dnd99 red

Anti-P. aeruginosa (ab68538) and anti-LAMP1 (ab24170) antibodies were obtained from AbCam. LysoTracker-DND99 Red, Alexa conjugated Annexin V, Rhodamine and Alexa-fluor 647 conjugated phalloidin, TO-PRO3 and CellTrace were acquired from Life Technologies. Cleaved Caspase-3 antibody (9661S) was obtained from Cell Signaling Technology. Purified Recombinant Annexin V was either purchased from BD Pharmingen or produced in the laboratory from pProEx.Htb.Annexin V plasmid (kindly provided by Dr. Seamus J. Martin)[63 (link)].

Human macrophages cultured in 24-well plates were treated as indicated above. In the last hour of infection, culture supernatants were replaced with medium that contained Lysotracker DND-99 Red (Life technologies) (100 nM). The slides were examined using a Zeiss Meta 510 laser confocal microscope with a plan-Apochromat 63× objective lens. A total of 10 fields containing 5–10 macrophages per field were examined in each experiment. The mean fluorescent intensity (MFI) for each macrophage was calculated using Image J software. Each cell from the image was selected and histogram analysis was performed. For immunostaining, mouse monoclonal antibodies for CD63 (sc-5275, Santa Cruz Biotechnology) and V1-ATPase H (sc-166227, santa Cruz Biotechnology) were visualized with anti-mouse-Alexafluor 588-conjugated secondary antibody. Goat polycolonal antibodies for active cathepsin D (sc-6486, Santa cruz) were visualized with anti-goat-Alexafluor 488-conjugated secondary antibody (Life technologies).