Toxoplasma gondii Inhibits Apoptosis in Infected Cells by Caspase Inactivation and NF-κB Activation T. gondii (3 × 106, cell:tachyzoite = 1:10) for 6 h in 6 well plates were visualized by TEM performed using standard methods. Briefly, spleen cells were fixed in 3% glutaraldehyde (PH 7.8) for 3 h at room temperature and post-fixed in 1% osmium tetroxide for 1.5 h at 4 . After serial dehydration with 50%, 70% and 100% ethanol, specimens were embedded in Epon resin. Ultrathin sections were loaded onto copper grids; stained with uranyl ace- tate and lead citrate, and visualized with a trans- mission electron microscope H-7600s (Hitachi, Tokyo, Japan). RESULTS DNA fragmentation in spleen cells was sup- pressed by T. gondii Spleen cells were incubated with AD and/or T. gondii tachyzoites for 5 h and 18 h respectively at 37 . DNA was then extracted from spleen cells, resolved on a 2% agarose gel, and stained with ethidium bromide. DNA laddering was observed in mouse spleen cells treated with AD for 5 h (data not shown) and 18 h (Fig. 1, lanes 1 & 2). Fig. 1. Fragmentation patterns of DNA from mouse spleen cells treated with actinomycin-D and/or T. gondii for 18 h. Fragmentation of DNA was down regulated in cells treated with AD/T. gondii (lane 6). Spleen cells infected with T. gondii exhibited only weak DNA fragmentation (lane 5). Lane 1. spleen cells treated with actinomycin-D (5 μg/mL); 2. spleen cells treated with actinomycin-D (10 μg/mL); 3. normal spleen cells for 18h incubation; 4. fresh normal spleen cells; 5. T. gondii infected spleen cells (cell:parasite = 1:10); 6. spleen cells infected with T. gondii and treated with actinomycin-D (5 μg/mL). In repeated experiments, similar results were shown. Apoptosis was down regulated in mouse spleen cells treated with AD and infected with T. gondii (Fig. 1, lane 6). Spleen cells infected with T. gondii only showed weak DNA fragmentation (Fig. 1, lane 5). DNA laddering was observed in spleen cells for 18h incubation, but not in freshly isolated cells (Fig. 1, lanes 3 & 4). Similar results were found upon repeated experiments. Inhibition of apoptosis in spleen cells infected with T. gondii was observed by flow cytometric analysis Mouse spleen cells treated with AD and/or T. gondii infection were pooled, incubated with annexin-FITC V and PI, and analyzed by flow cytometry. Early apoptotic cells have increased annexin V, but not PI staining. Late apoptotic or necrotic cells stain with both annexin V and PI. Mouse spleen cells cultured with AD, AD/T. Fig. 2. Flow cytometric analysis of apoptosis in mouse spleen cells treated with actinomycin-D and/or T. gondii for 18 h. (%) indicates level of apoptosis. (A) actinomycin- D (5 μg/mL), 80.3%; (B) actinomycin-D (10 μg/mL), 81.2%; (C) normal spleen cells for 18h incubation, 39.6%; (D) freshly isolated spleen cells, 23.8%; (E) T. gondii infected, 18.2%; (F) T. gondii and actinomycin-D treated spleen cells, 30.1%. Flow cytometry was repeated more than three times and similar results were observed. Yonsei Med J Vol. 47, No. 6, 2006