TLR2 activity of <i>S</i>. <i>aureus</i> and <i>B</i>. <i>anthracis</i> PGNs.

<p><i>B</i>. <i>anthracis</i> (A) and <i>S</i>. <i>aureus</i> (B) PGN samples were taken after the indicated steps of the purification process. The samples were washed three times with endotoxin free (LAL) water, dried, weighed and tested for TLR2 activity at a concentration of 10 μg/ml, using the HEK-TLR2 cell assay. The data are expressed as the mean ± SEM fold difference TLR2 activity over the negative control, endotoxin free water. Statistical significance was determined by ANOVA with Bonferroni post test. *, **p<0.01, ***p<0.001 <i>versus</i> endotoxin free water. C & D: Freshly prepared peripheral blood mononuclear cells were stimulated for 12 hours with 10 μg/mL <i>B</i>. <i>anthracis</i> (Panel C) or <i>S</i>. <i>aureus</i> (Panel D) PGN samples taken at defined steps during the purification procedure, as described in Panels A & B. The cells were stained for intracellular TNFα and then analyzed by flow cytometry. The internalization requirement was assessed in the presence of 15 μM cytochalasin D (CytoD). Data are expressed as the mean ± SEM of TNFα+CD14+ cells. Statistical significance was determined by paired two-way ANOVA with Bonferroni post hoc test for multiple comparisons. *p<0.05, **p<0.01, ***p<0.001, ns p>0.05.