Listerial strains were grown in an overnight culture of Brain Heart Infusion (BHI) medium with shaking at 37°C. The next morning, bacterial cultures were diluted 1:10 and see more grown in BHI broth at 37°C until mid-log phase was reached. Bacteria were then harvested by centrifugation, washed GS-9973 molecular weight several times and resuspended in sterile PBS. The numbers of colony forming units (CFU) of L. monocytogenes
were determined by counting cells in a THOMA-chamber and by calculating the appropriate number of bacteria for infection. Plating bacteria on BHI agar plates verified the actual number of CFU in the inoculum. Animal infection Age matched groups of female mice (10-12 weeks), were prepared for infection challenge by withheld of food for 12 h; drinking water was replaced by carbonate buffered water (2,6% NaHCO3). Bacteria were prepared as described . Briefly, a total of 0.2 MK0683 chemical structure ml of the desired inoculum of either strain was mixed with 0.3 ml PBS containing 50 mg CaCO3. A suspension of 5 × 109 CFU was inoculated intragastrically into mice using a 21-gauge feeding needle attached to a 1 ml syringe. After infection mice were given access
to food and water ad libitum. For CFU determination, small intestines, mesenteric lymph nodes, spleens, livers, gallbladders and brain of sacrificed mice were aseptically removed. To determine only intracellular bacterial load in small intestines, organs were washed with PBS and incubated in DMEM containing 100 μg/ml gentamicin for 2 h to kill extracellular bacteria. Serial dilutions of homogenates were plated on BHI agar plates and colonies were counted after overnight incubation at 37°C. All samples were weighted and homogenized in pre-cooled PBS. For histopathological analysis of liver and spleen, organs were fixed in 10% buffered formalin, dehydrated, and embedded in paraffin. Sections of 4 μm were cut and stained with hematoxylin-eosin (H&E), and assessed
blind by one researcher (PB) for evaluation of pathologic changes. In vivo imaging For detection of bioluminescence, mice were anesthetized using isoflurane (Abbott Animal Health). Isoflurane gas anesthetic was administered at 2% in oxygen, which enables mild anaesthesia. BLI images were obtained using cAMP an IVIS 200 imaging system (CaliperLS) with integration time of 4 min at a binning of 8 and F/stop of 1. For the detection of in vivo enzymatic activity of the firefly luciferase, IFN-β-reporter mice were injected intravenously (i.v.) with 150 mg/kg of D-Luciferin (Synchem) in PBS, 5-10 min prior to imaging. Mice were anesthetized with isoflurane and monitored using the IVIS 200 imaging system according to manufactures instructions. Camera settings and exposure time were identical for all images. Photon flux was quantified by using the Living Image 3.1 software (CaliperLS).