Specific murine IgG2a isotype controls were used to monitor non-specific binding. Stained peritoneal cells were washed

with PBS containing 2% fetal bovine serum (FBS), pelleted by centrifugation at 400 × g and fixed with PBS containing 1% (w/v) paraformaldehyde. A total of 30,000 events were acquired (FACSCanto™; Becton Dickinson, CA, USA) using the FACS Diva software (version 6.1.3) for data acquisition and analysis. Data were expressed as the mean ± SEM. Statistical variations were analyzed using multi-factorial ANOVA. P < 0.05 was considered statistically I-BET-762 mw significant. The response of mice to the i.p. injection of Ts2 or Ts6 was studied by evaluating the influx of leukocytes into the peritoneal cavity. Ts2 or Ts6 i.p. inoculation in mice induced an increase of total leukocyte (Fig. 1A) and neutrophil (Fig. 1B) numbers Alectinib purchase in the peritoneal cavity throughout the experimental time course (4, 24, 48 and 96 h). The mononuclear cells were increased after 4 and 96 h post Ts2 injection compared to mice inoculated with PBS. Ts6 increased

mononuclear cells only after 96 h (Fig. 1C). We also determined the acute phase protein levels in the peritoneal fluid of mice injected with Ts2 or Ts6 (Fig. 2). Compared to control, the total protein levels of the peritoneal fluid peaked between 24 and 48 h and then decreased at 96 h after injection with Ts2 or Ts6 (Fig. 2). Taken together, these results demonstrated that Ts2 or Ts6 induced an inflammatory response in the peritoneal cavity, mainly during the first 24 h. Fig. 3 shows the profile of inflammatory cytokines released in the peritoneal cavity after the injection with Ts2 or Ts6. We demonstrated that Ts2 and Ts6 altered the release of specific cytokines in a time-dependent manner. Ts2 augmented the release of IL-6, IFN-γ and the regulatory cytokine IL-10 at 4 h (Fig. 3A, D and E, respectively); 17-DMAG (Alvespimycin) HCl at 24 h, however, only IL-10 was increased (Fig. 3E). At 48 h post Ts2 injection, there was an increase in the levels of TNF-α, IFN-γ, IL-10 and IL-4 (Fig. 3B, D, E and F, respectively), while at 96 h, we only observed an increase in TNF-α, IL-1β and IL-10 (Fig. 3B,

C and E, respectively). Additionally, we observed a mild difference in the cytokine profile following the Ts6 injection compared to Ts2. After 4 h, Ts6 increased the release of IL-6, TNF-α, IL-1β and IFN-γ (Fig. 3A–D, respectively), while only the release of IFN-γ was increased at 24 h (Fig. 3D). At 48 h, TNF-α and IFN-γ levels were increased (Fig. 3B and D), while only TNF-α was increased after 96 h (Fig. 3B). For comparison, the changes in the corresponding cytokine release were also measured in a control group of mice that received PBS injection. To determine whether LTB4 and PGE2 are produced as a result of the toxin injection, groups of mice were i.p. inoculated with Ts2 or Ts6 and the peritoneal fluid was collected after 4, 24, 48 and 96 h.