Las x sp8

Fixed samples were analyzed with a confocal laser scanning microscope (Leica TCS SP8) equipped with a 63x, NA1.4 oil immersion objective and Leica LAS X SP8 software (Leica Microsystems) was used for acquisition.

Fixed samples were analyzed with a confocal laser scanning microscope (Leica TCS SP8) equipped with a 63x oil immersion objective (NA 1.4) and Leica LAS X SP8 software (Leica Microsystems, Wetzlar, Germany) was used for acquisition.

One day before infection 2.5 x 10⁴ HeLa cells were seeded in ibidi μ-Slide 8 wells. The following day cells were infected with different bacterial strains as indicated and after 30 min the medium was replaced by 200 μl CCF4/AM loading solution (prepared according to the manufacturer’s instructions) diluted in DMEM supplemented with 10% FCS and 2.5 mM probenecid. The cells were placed in the prewarmed chamber supplied with 5% CO2 of the laser scanning microscope Leica TCS SP8 and imaging was performed using a 20x oil immersion objective (NA 0.75) and the Leica LAS X SP8 software (Leica Microsystems, Wetzlar, Germany).

For analysis of bacterial cluster size and size distribution, bacteria were grown statically for 18 h at 37°C in TSB and ASF, respectively. Thirty minutes prior to staining, the bacterial sediment was carefully dispersed by vortexing, and 10 µl were transferred to staining buffer (3% BSA in PBS + DAPI 300 nM, Invitrogen) in a µ-Slide 8-Well (ibidi, Gräfelfing, Germany). Samples were analyzed with the confocal laser scanning microscope Leica TCS SP8 equipped with a ×63, NA1.4 oil immersion objective and LAS X SP8 software (Leica Microsystems, Wetzlar, Germany). At least 10 positions per condition were recorded as stacks with a distance of 500 nm and 2,048 × 2,048 pixels. Bacterial detection and segmentation were done using the Imaris software package (Oxford Instruments, Abingdon, UK). Subsequently, cluster analysis was done in MatLab (Version 9.2, The MathWorks Inc., Natick, MA, USA). In brief, the position of each bacterium was calculated by its volume, and the center of mass of the segmented volume was defined. Distances between centers of masses were measured and categorized into clusters with a threshold of ≥5 bacteria/cluster. The MatLab script used is available in MatLab Supplement S1.

For structural cytoskeleton analysis, U‐118 MG cells were cultured on microscope coverslips and transfected under standard conditions as described previously. 24 h post‐transfection, cells were fixed with the use of Image‐iT™ Fixation/Permeabilization Kit (Invitrogen) according to the manufacturer’s protocol. F‐actin fibres were visualized by phalloidin conjugated to tetramethylrhodamine (Invitrogen) with simultaneous use of DAPI (Sigma‐Aldrich) to visualize cell nuclei. Staining was performed according to the manufacturer’s protocol. Pictures were obtained with the use of Leica TCS SP5 confocal microscope and software LAS X SP8 (Leica).