Figure 5.

Analysis of caspase-3 activity in neutrophils and effect of caspase inhibitors. (a) Activation of caspase-3 was determined by detection of chromophore p-nitroanilide (pNA) after cleavage of the labelled substrate DEVD-pNA. After co-cultivation of neutrophils with Trichomonas vaginalis for 24 h, cell lysate supernatants were prepared at the designated times. The supernatants were incubated with DEVD-pNA for 1 h at 37°C. Optical density was measured at 405 nm and presented as the ratio of T. vaginalis-treated neutrophils/untreated neutrophils. *P < 0·05; significant difference from control group. (b) Effect of inhibitors of caspase-3 and caspase-8 on spontaneous and T. vaginalis-induced neutrophil apoptosis. Neutrophils (1 × 10⁶/mL) were pre-treated for 10 min at 37°C with 25 mm zDEVD-fmk or zIETD-fmk to inhibit caspase-3 and 8, respectively. The cells were subsequently incubated with T. vaginalis (neutrophils/T. vaginalis, 10: 1) or in medium alone and then cultured for 12 h. Untreated cells (no inhibitors) were cultured with or without trichomonads for the same period of time. The percentage of apoptotic cells was obtained by flow cytometry using DiOC₆. The data represent mean ± SEM of four separate experiments. (*, significantly different from cells not exposed to inhibitors; P < 0·05)