GFP-expressing S. aureus (Newman strain) were harvested during their logarithmic-growth phase and incubated with BMDMs at an MOI of 10 for 60 and 120 min. LysoTracker-Red (Invitrogen) was added to BMDMs 30 min before the end of the incubation period and extracellular S. aureus were lysed with Lysostaphin (Sigma). Cells were fixed with 4% PFA in PBS and confocal images were acquired with an inverted Zeiss LSM510 laser scanning confocal microscope and a 60x NA 1.45 oil-immersion objective (Zeiss). Image analysis was done using ImageJ software (NIH). The percentage of phagocytosed S. aureus colocalizing with LysoTracker-Red was determined by blinded counting. To study trafficking of phagocytosed mycobacteria to lysosomes, BMDMs were infected with 10 MOI of GFP-expressing Bacille Calmette-Guerin (BCG, an attenuated strain of M. bovis) (45 (link)) for 3 h, then washed 3 times with media and incubated another 24 h in RPMI medium at 37°C. Cells were washed once and fixed with 4% PFA in PBS for 30 min, permeabilized with 0.1% saponin in PBS for 5 min and incubated with polyclonal rabbit anti-LAMP1 antibody (Abcam). Images were captured using a Nikon Eclipse TiE/B automated fluorescent microscope with Photometrics HQ@ Monochrome digital camera. 60x z-stack images were acquired and analyzed using NIS-Elements DUO software as previously described (45 (link)).