Automated Fluorescent Confocal Microscopy Assays

Images were acquired using an automated fluorescent confocal microscope (In Cell analyzer 6000, GE) equipped with a 20X (NA 0.70) air lens or 60X (NA 1.2) water lens for Tirap localization, intracellular mycobacterial replication, phagosomal acidification, phagosomal rupture, LD formation and quantitation of intracellular Mtb secreted effectors assays. The confocal microscope was equipped with 405, 488, 561 and 642 nm excitation lasers. The emitted fluorescence was captured using a camera associated with a set of filters covering a detection wavelength ranging from 450 to 690 nm. Hoechst 33342-stained nuclei and CCF4-stained cells were detected using the 405 nm laser with a 450/50-nm emission filter. Green signals corresponding to LysoTracker Green DND-26, CCF4-AM, ZsGreen⁺ Tcells, and H37Rv-GFP were recorded using 488 nm laser with 540/75-nm emission filters. Red signals corresponding to H37Rv-DsRed was recorded using 561 nm laser with 600/40-nm emission filters. LipidTOX signal was detected using 630-nm excitation and 690-nm emission wavelengths.