Axiovert z1

A375 melanoma cells (ATCC) were seeded in LabTek glass chamber slides (Nunc) at a density of 2 × 10⁴ cells per well. Cells were washed in PBS and fixed in 4% formaldehyde PBS for 15 minutes and then incubated with PBS-T, 10% goat serum, 0.05% Tween for 25 minutes at room temperature. Anti-CSPG4 IgG₁/IgE or control NIP antibodies were incubated for 45 minutes with A375 cells at a concentration of 10 μg/mL. Cell-bound antibodies were detected with a secondary goat anti-human IgG-FITC (Jackson ImmunoResearch) or goat anti-human IgE antibody conjugated to FITC (Vector Labs). Nuclei were stained in Hoechst dye for 3 minutes. All washing steps used PBS, except the final wash with dH₂0. Cells were then mounted in Mowiol (Sigma) mounting medium. Fluorescence microscopy was performed on a Zeiss Axiovert Z.1 (40× objective) upright microscope. AxioCamMR3 and AxioVision Software (Carl Zeiss) was used for acquisition and analysis.

To monitor Wnt/β-catenin transcriptional activity, we transfected THP1 with reporter plasmids encoding for an inducible TCF responsive GFP reporter (Qiagen, Hilden, Germany) as previously described [8 (link)] GFP signal was observed through Axiovert Z1 (Zeiss, Sheung Kehen, Germany) and quantitatively measured by flow cytometry. The Wnt pathway activity was determined by normalizing the activity of TCF-GFP to that of CMV-GFP plasmid.

The induction assays have been described previously (8 (link)). Briefly, bacteria were resuspended in prewarmed DMEM containing 1% FBS, filtered through 5-μm filters, and diluted to 10⁷ CFU/ml. The bacterial solutions were transferred to 24-well glass-bottom plates (MatTek) and allowed to grow and form microcolonies for 3 h at 32°C or 37°C in a 5% CO₂ environment in a live-cell observer (Axiovert Z1; Zeiss). Prewarmed DMEM supplemented with sodium l-lactate (L7022; Sigma-Aldrich) was added to microcolonies at a 1:1 ratio at final concentrations of 10 mM. During treatment with spectinomycin, the antibiotic was added at a final concentration of 100 μg/ml 30 min before the induction. Three positions were chosen per well, and images were acquired every 5 to 10 min for 4 or 5 h using a 40× objective. The aggregation phase is described as the start of incubation until the dispersal of bacteria starts. The dispersal phase is described as the initiation of bacterial dispersal until no aggregates are observed. The planktonic phase is described as dispersed single bacteria.

N. meningitidis FAM20 was resuspended (10⁷ CFU/ml) in prewarmed medium containing 1% FBS and filtered through a 5-μm pore filter to break apart preexisting bacterial aggregates. The bacteria were incubated in 24-well glass-bottom plates (MatTek) in 1 ml at 37°C in a 95% air/5% CO₂. At 1.5 h, the oxygen concentration was set to 0%. Oxygen depletion was conducted by a 95% N₂/5% CO₂ flow. At 5 h, the oxygen concentration was set to 95% air/5% CO₂. The bacteria were observed under a live-cell microscope (Axiovert Z1, Zeiss). Three images per well were acquired every 10 min for 8 h using a 40× objective.

After infection, HeLa cells were fixed by plunge-freezing using a Vitrobot Mark IV (Thermo Fisher Scientific). Prior to vitrification, samples were slightly fixed with 1% PFA for 5 min to inactivate bacteria and imaged using an Axiovert Z1 driven by ZEN Blue 2.3 software (Carl Zeiss) epi-fluorescence microscope or a confocal microscope LSM710 (Carl Zeiss) driven by ZEN 2010 software. To stain for mitochondria, samples were incubated with 100 nM of Mitotracker Red (Invitrogen) for 30 min before vitrification. In all cases, grids were incubated with 100 nm fiducial gold nanoparticles (that would help during the alignment of the tilt series) for 30 s before vitrification. Following vitrification, grids were shipped to the Mistral beamline at the ALBA synchrotron (Barcelona, Spain). Vitrified grids were then transferred in liquid nitrogen to the cryo-correlative cooling stage Linkam CSM196 (Linkam Scientific Instruments) to hold samples at a stable −190°C during analysis. The cryo-stage was inserted into an AxioScope A1 (Carl Zeiss) epifluorescence microscope with a N-Achroplan 50×/0.6 Ph1 objective and imaged with a CCD AxioCam ICm1 (Carl Zeiss).
Cryo-fluorescence microscopy was used to pre-select vitrified samples and map the position of cells. Selected samples were then transferred to the Mistral synchrotron beamline at liquid nitrogen temperature.

N. meningitidis FAM20 and its isogenic mutants were resuspended in prewarmed DMEM containing 1% FBS, filtered through 5-μm filters to break existing aggregates, and diluted to preferred bacterial concentrations (2 × 10⁷, 1 × 10⁷, 8 × 10⁶, 6 × 10⁶, 4 × 10⁶, and 2 × 10⁶) in the presence of 2 mM l-lactate (L7022; Sigma-Aldrich). Microcolony dispersal was observed using time-lapse microscopy (Axiovert Z1; Zeiss). Three positions were chosen per well, and images were acquired every 10 min for 6 h using a 40× objective. The images were independently evaluated by visual inspection. The aggregation phase was defined as the time from the start of incubation until dispersal of bacteria starts (i.e., when the microcolonies reached maximal size and started to decrease size). The dispersal phase was defined as the time from initiation of bacterial dispersal until microcolonies were no longer visible in the images. The planktonic phase was described as dispersed single bacteria. For wild-type strain and 5 mM lactate, the dispersal was rapid and obvious and occurred from one image to another, i.e., within a 10-min time frame.