Analyzing Mtb Infection in Macrophages

siRNA-transfected RAW cells or BMDMs were infected with GFP-expressing Mtb for 4 h as described above. One day before infection, macrophages were treated with IFN-γ (100 U ml⁻¹) or left untreated. Cells were fixed with 1% PFA in PBS overnight at 24 h postinfection, permeabilized with 0.1% saponin in PBS and blocked with 2% BSA in PBS for 1 h. Cells were then immunostained for LAMP1 (1:1000 dilution; Abcam) for at least 1 h at room temperature or at 4 °C overnight, followed by treatment with anti-rabbit Alexa Fluor 594-conjugated secondary antibody (Invitrogen). Images were captured using a Nikon Eclipse TiE/B automated fluorescent microscope described above and analysed using NIS-Elements DUO software as described previously^{20 (link)}.