Metavue software

Digital images of the plaques were obtained using AxioObserver.Z1 microscope (Zeiss, Jena, Germany). Plaque area and thickness were measured using ImageJ software. Plaque area was calculated by measuring the surface delimited by the internal elastic lamina and distracting the surface of the lumen. The results were normalized to total vessel cross-sectional area for each arterial section to eliminate variations due solely to vessel size. The minimum fibrous cap thickness at each plaque section and the media thickness at the thickest region of the plaque were measured for each section. Intima–media thickness (IMT) was calculated.
To analyze the macrophage load in the plaques, the color threshold for immunostained cells was manually adjusted in the images until the computerized detection matched the visual interpretation (MetaVue Software; Molecular Devices LLC, San Jose, CA, USA). The numbers of immunoreactive cells were additionally digitally counted (NIS-Elements; Nikon Instruments, Düsseldorf, Germany) in the representative areas of 0.2 mm² in the plaque shoulders, lipid core, and fibrous cap. Analyses were carried out by two independent observers (BL, IC). The intra- and interobserver variability was lower than 10%.

For the kinetic assays, strains containing the relevant plasmids were grown overnight in BHI. The cultures were then diluted to 1/100 in BHI, then grown in 96-well microtiter plates for 135 min at 37°C until the exponential growth phase (14 (link)). To induce expression of SprG1, SprG2, SprG3 and SprG4, the cultures were then incubated with 1 or 2 μM of aTc. For the kinetics, the optical absorbance at 600 nm (OD₆₀₀) was measured at 30-min intervals using a Synergy 2 multi-mode reader (Biotek). To determine the effects of SprG induction on bacterial growth, cultures were prepared by 2-fold serial dilutions of exponential phase cultures on BHI plates containing 1 or 2 μM aTc, then incubated for 24 h at 37°C. For the cell death experiments, cultures were incubated for 135 min, washed with 9‰ saline solution, then stained with a LIVE/DEAD bacterial viability kit (Invitrogen). Pictures of fluorescence-labeled cells were captured with a Leica DM RXA2 microscope and a CoolSNAP HQ charge-coupled device camera using Metavue software (Molecular Devices).