Brucella Localization in Lysosomes

RAW 264.7 cells were cultured on 15-mm glass coverslips (Thermo Scientific, Waltham, MA, USA) in 24-well plates and infected with the WT strain or the Δ27735 mutant at an MOI of 200, as described above. At 4 and 24 h pi, the cells were washed twice with PBS and fixed overnight in 4% (w/v) paraformaldehyde at 4 °C. Fluorescence staining and a Brucella co-localization assay with lysosomes were performed as described in our previous report [11 (link)]. Rabbit anti-Brucella polyclonal antibody (1:500 dilution) was used to track intracellular bacteria. Rat LAMP-1 (lysosome associated membrane protein 1) monoclonal antibody (1:1000 dilution; Abcam, Cambridge, UK) was used to track the lysosomes. Goat anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 (Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies at dilutions of 1:1000. The cells were observed under laser scanning confocal microscope (Nikon D-Eclipse C1, Tokyo, Japan) with 100× oil immersion objective. Images were saved in TIFF format and imported to Adobe Photoshop CS4 (Adobe Systems Incorporated, San Jose, CA, USA), where they were merged using RGB format. To determine the percentage of bacteria positive for the lysosome marker LAMP-1, 100 intracellular bacteria were counted randomly. Assays were performed in triplicate.