Quantifying Brucella Invasion in Macrophages

Macrophages were infected as described above with Brucella–GFP (MOI of 100:1) and the bacteria number was assessed in cells infected for 24 h, as previously described [47 (link)]. Cells were washed twice with PBS and fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 30 min. After fixation, coverslips were washed three times with PBS. Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen, Carlsbad, California, USA). Confocal microscopy analysis was performed in a Nikon A1 confocal system. Three coverslips were analyzed per sample and images were taken using a 10× objective for six random areas of each coverslip. Images presented here are representative.