, Long Beach, CA) or an anti-HA.11 mAb (1:1000; Covance) for 1 h at room temperature. After washing three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (1:1000; Amersham Pharmacia Biotech, Piscataway, NJ) diluted in PBS-SM, for 1 h at 37°C. After washing three times, the proteins were visualized on X-ray film using ECL™ western blotting detection reagents (GE Healthcare

UK Ltd., this website Buckinghamshire, UK) according to the manufacturer’s recommendations. Selleck PRN1371 Parasite infections in mice Parasites purified from in vitro cultures were washed in sterile PBS and tachyzoites (5 × 102 – 1 × 103) were inoculated intraperitoneally into mice. Three or five days after the infection, cells were collected from the peritoneal cavity of naïve or parasite-infected mice by peritoneal washing with 5 ml of cold PBS. After harvesting, the cells were centrifuged at 800 × g for 10 min and suspended in cold PBS. These cells were then subjected to flow cytometry. Supernatants were used to measure TgCyp18, IL-12, CCL2, CCL5 and CXCL10 production. To determine the parasite burden and chemokine expression levels in the mice, tissues including the brain, liver, lungs

and spleen from T. gondii infected and uninfected animals were collected at 0, 3 and 5 days post-infection (dpi). Sandwich enzyme-linked GSK126 purchase immunosorbent assay (ELISA) detection of TgCyp18 The presence of TgCyp18 in mouse ascites fluid and TgCyp18 secreted by extracellular parasites in infected mice was determined by a sandwich ELISA as described previously [14]. To detect TgCyp18 from extracellular tachyzoites, purified

T. gondii tachyzoites (3 × 107) were incubated in 1.5 ml of GIT medium (Nihon Pharmaceutical Co., Ltd, Tokyo, Japan) at 37°C. Before transferring parasite suspensions MTMR9 from ice to 37°C for a secretion assay, 250 μl of the parasite suspension was removed and processed as the time zero reading. The remainder of the parasite suspension was incubated at 37°C in a water bath. After 15, 30, 60, and 120 min, 250 μl of parasite suspension was removed. The culture supernatants were centrifuged (760 × g for 10 min at 4°C, then 7000 × g for 10 min at 4°C) together with the ascites fluid from the in vivo experiment, and then subjected to sandwich ELISA. Microtiter plates were coated with 1 μg of rabbit anti-rTgCyp18 polyclonal IgG [13] diluted in 0.05 M carbonate buffer (pH 9.6), which was used as the capture antibody at 4°C overnight. Blocking was performed with a blocking solution (PBS-SM, pH 7.2) at 37°C for 2 h. Microtiter plates were incubated at 37°C for 30 min with each supernatant in triplicate. After washing six times with PBS-T, anti-TgCyp18 mouse serum (1:100) was added to each well as the detection antibody.