During early development, germ cells are set aside from the somatic cells of the embryo on the basis of differential regulation of mRNA in germ cells compared to somatic cells

curve analysis was performed according to the manufacturer’s instructions; PCR primer efficiencies were as follows: 1.92 for IL-6, 1.8 for IL-8, 1.83 for CXCL1, 1.99 for CXCL2, 1.94 for CCL20 and 1.88 for GAPDH. Calculation of relative gene expression included adjustments for PCR efficiencies and using the following equation: Relative gene expression = target gene efficiency6/1.886. solution were used as negative and positive control for neutrophil recruitment, respectively. After 4 hours, cells were harvested from the peritoneal cavity in PBS 0.2% BSA. One-hundred ml of cell suspension was directly stained with FITC-labeled rat anti-mouse Gr-1 monoclonal antibody or the appropriate isotype control for 30 min at 4uC and analyzed by flow cytometry. The flow cytometer was set to count events during a fixed time thus permitting quantification of the absolute number of recovered Gr-1 positive cells in each mouse. A quality check was performed on the flow cytometer before use to assure a constant flow rate. GLPG0634 site Myeloperoxidase assay Skin samples of mice were homogenized in 500 ml 0.05% hexadecyltrimethylammonium bromide solution. Homogenates were centrifuged for at 18,0006 g for 30 min at 4uC. Supernatants were transferred to a clean microcentrifuge tube and stored at 280uC until further analysis. Next, 10 mg of o-dianisidine dihydrochloride was added to 60 ml of freshly-prepared HTAB solution to yield DCC solution. In addition, activated substrate was prepared by adding one ml of 0.05% hydrogen peroxide solution for every 99 ml of DCC solution. Finally, the reaction was started by adding 90 ul of DCC solution in HTAB solution and 100 ml of activated solution to 10 ml of skin supernatants 96 well flat-bottom plates. The absorbance was read every 15647369 minute for 10 minutes at 450 nm using a spectrophotometer. All samples were analyzed in triplicate. For quantification purposes, a calibration curve of horseradish peroxidase ranging from 100 mU/ml to 3.13 mU/ml was run in parallel with the samples in triplicate with every experiment. 21804608 Chemokine secretion in hBMEC supernatants HBMEC supernatants were collected after infection with B. anthracis Sterne, DpXO1, DLF, DEF, or DLF/EF deletion mutants after 6 hours. Concentrations of IL-8, CXCL1, CXCL2 and CCL20 were measured using enzyme-linked immunosorbent assays according to the manufacturer’s instructions. IL-6 and IL-8 concentrations were measured using the cytometric bead array system according to the manufacturer’s instructions. Statistical analysis Graphpad Prism version 4.03 was used for statistical analysis. Differences in adherence/invasion, mRNA expression, chemokine secretion in hBMEC supernatants were evaluated with a one-way ANOVA followed by Tukey’s post hoc test. Differences in neutrophil recruitment were determined using a paired t-test for the MPO assay and an unpaired t-test for the intraperitoneal infection model. Kaplan-Meier survival plots were evaluated with the log-rank test. Statistical significance was accepted at p,0.05. Mouse infection studies All animal experiments were approved by the Committee on the Use and Care of Animals, and performed using accepted veterinary standards. For the meningitis model, bacteria were grown to early log phase, washed in PBS and resuspended to an optical density of 0.4 in PBS. Vegetative bacteria were diluted in PBS to 236105 CFU/ml and 0.1 ml was injected intravenously into 8 weeks old out bred immunocompetent female CD-1 mice. Mice were monitored f

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