Visualizing Brucella Infection and STING Activation in MEFs

C57BL/6 or STING KO mouse embryonic fibroblasts (MEFs) (1×10⁴) were plated onto 24 wells containing glass coverslips and infected as described above with Brucella-GFP. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Cells were subsequently blocked for 1 h with 3% BSA in PBS at room temperature prior to incubation with anti–GRP78 (BiP) primary antibody (Abcam, Cambridge, UK) at 4 °C overnight. For evaluation of STING activation by B. abortus, MEFs cells were infected with B. abortus (MOI of 1000:1) or transfected with dsDNA90 (3 μg/ml) for 4 h. Cells were processed for immunofluorescence as described above and incubated with a rabbit polyclonal antibody against STING as described previously (10 (link)). Anti-rabbit conjugated with Alexa Fluor 546 was used for detection of primary antibodies. Coverslips were mounted in slides using ProLong Gold with DAPI mounting medium (Invitrogen) and confocal microscopy analysis was performed in a Nikon A1 confocal system. Confocal microscopy analysis was performed 4 h after infection given that at this time-point, BiP-expression and STING activation were enhanced on MEFs cells. Three coverslips were analyzed per sample and representative images were taken using a ×40 objective.