FIG. 4.

The cytotoxic activities in S. flexneri culture supernatants. S. flexneri EP and sBLP coelute from an RP-HPLC column. Eight hundred microliters of a phenol extract of S. flexneri culture supernatants (A), 200 μg of EP (B), or 200 μg of sBLP (C) was loaded onto a C₄ RP-HPLC column and eluted with a linear gradient of increasing ACN concentration. One-milliliter fractions were collected and assayed for cytotoxic activity by an LDH release assay on THP-1 cells by serial dilution directly into tissue culture media. Activity is defined as 1/(dilution yielding 50% maximal cytotoxic activity). HPLC conditions were as follows. Buffer A contained dH₂O and 0.1% TFA, buffer B contained 95% ACN and 0.1% TFA, and the flow rate was 1 ml/min. The gradient was as follows: 0 to 15 min, 0 to 100% buffer B (linear gradient); 15 to 17 min, 100% buffer B; 17 to 19 min, 100 to 0% buffer B.