FIG. 3.

(A) Purification of commercially available S. aureus LTA. LTA was purified with an octyl-Sepharose column as described in Materials and Methods. The column was successfully eluted with a linear gradient of 15 to 80% propan-1-ol in equilibration buffer (dashed line). Fractions of 2 ml were collected, dialyzed, lyophilized, and resuspended in endotoxin-free water and tested for phosphate content as described in Materials and Methods. The same fractions were also subjected to an LAL assay to test endotoxin levels either in the absence (solid triangles) or the presence (open triangles) of 10 μg of polymyxin B (PMB)/ml. Data represent averages of duplicate samples. Experiments were repeated two times. (B) Induction of NO by eluted LTA fractions. Macrophages were stimulated with different LTA fractions (1:33 dilution in culture medium) for 20 h in either the absence (solid circles) or the presence (open circles) of 10 μg of polymyxin B/ml before supernatants were harvested for a nitrite assay as described in Materials and Methods. Data represent averages of triplicate samples. Experiments were repeated two times under equivalent conditions.