LysoTracker

Cells, grown on coverslips, were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) and quenched with 50 mM NH₄Cl. Then, cells were permeabilised with 0.2% Triton X-100 for 5 or 7 min (depending on the antibody) and blocked for 30 min in PBS containing 10% FBS and 1% bovine serum albumin (BSA). Primary antibodies were detected with Alexa Fluor-conjugated secondary antibodies.
For lysosome staining, cells were incubated for 1 h with Lysotracker (1:1000) in complete medium before fixing. Images were collected using a laser scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with a planapo 63× oil-immersion (NA 1.4) objective lens by using the appropriate laser lines. Images were acquired with the confocal pinhole set to one Airy unit, taking Z-slices from the top to the bottom of the cell by using the same setting (laser power, detector gain), as well as the same threshold of fluorescence intensity in all experimental conditions (control and silenced cells). Quantification and colocalisation analyses were carried out using LSM 510 software as previously described^{58 (link),59 (link)}. The mean fluorescence intensities were measured by drawing regions of interest (ROIs) around the entire cell and corrected for background. The number and size of fluorescent puncta were carried out by using ImageJ software^{59 (link)}. For GFP–EGFR internalisation experiments, the mean fluorescence intensity of surface and intracellular GFP signals was measured by drawing ROIs around plasma membrane labelled by a specific marker (CD55) and around areas, which excluded surface signals, respectively.

For tracking of lysosomes and mitochondria, cells were double-stained live with 50 nM Lysotracker Red DND-99 (Molecular Probes Cat. No. L-7528) and 50 nM Mitotracker Deep Red FM (Molecular Probes Cat. No. M22426). For measuring mitochondrial membrane potential (and tracking as well), cells were stained with 200 nM Mitotracker JC-1 (Molecular Probes Cat. No. M34152). Trackers were added directly to culture supernatants and incubated for 1 h at 37 °C. Imaging was then performed without further washing of cells. Live imaging of compartmentalized axons in Xona Microfluidic Chambers (MFC) was performed with a Leica HC PL APO 100 × 1.46 oil immersion objective on an inversed fluorescent Leica DMI6000 microscope enclosed in an incubator chamber (37 °C, 5% CO₂, humid air) and fitted with a 12-bit Andor iXON 897 EMCCD camera (512×512, 16 µm pixels, 229.55 nm/pixel at 100Χ magnification). For more details, refer to https://www.biodip.de/wiki/Bioz06_-_Leica_AFLX6000_TIRF. Excitation was performed with a TIRF Laser module in epifluorescence (widefield) mode with lines at 488, 561 and 633 nm. Fast dual color movies were recorded at 3.3 frames per second (fps) per channel over 2 min (400 frames in total per channel) with 115 ms exposure time as follows: Lysotracker Red (excitation: 561 nm, emission filter TRITC 605/65 nm) and Mitotracker Deep Red (excitation: 633 nm, emission filter Cy5 720/60 nm) or for Mitotracker JC-1 with excitation at 488 nm and fast switching between emission filter FITC 527/30 nm (green channel for compromised membrane potential) and TRITC 605/65 nm (red channel for intact membrane potential). Movie acquisition was performed at strictly standardized readout positions within the micro channels of the micro groove barrier that separated the proximal seeding site from the distal axonal exit as illustrated in Fig. 2a. Specifically, the readout windows were located either just adjacent to the channel exit (distal readout) or the channel entry (proximal readout).

Images from the automated confocal microscope were analyzed using multi-parameter scripts developed using Columbus system (version 2.3.1; PerkinElmer). Segmentation algorithms were applied to input images to detect nuclei and the signal of Mtb H37Rv to determine infection and replication rates. Briefly, the host cell segmentation was performed using two different Hoechst signal intensities—a strong intensity corresponding to the nucleus and a weak intensity in cytoplasm—with the algorithm “Find Nuclei” and “Find Cytoplasm”, as described previously [25 (link)]. GFP or DsRed signal intensities in a cell were used for the intracellular bacterial segmentation with the algorithm “Find Spots”. The identified intracellular bacteria were quantified as intracellular Mtb area with number of pixels. Subsequently, the population of infected cells was determined, and the increase of intracellular Mtb area, corresponding to intracellular mycobacterial replication, was calculated. For quantification of phagosomal acidification with Lysotracker Green DND-26, green signal intensity in a cell was used for the intracellular acidic compartment segmentation with the algorithm “Find Spots”.

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.