Toxoplasma gondii Inhibits Apoptosis in Infected Cells by Caspase Inactivation and NF-κB Activation strain) results in an over production of IFN-γ and high levels of apoptosis.8 Conversely, some intra- cellular protozoa have the ability to inhibit host cell apoptotic responses. T. gondii activates a survival response in infected cells by increasing host resistance to multiple apoptotic stimuli.9 In the present study, we focus on the inhibition of apoptosis in mouse spleen cells infected with T. gondii using morphological, molecular, and bio- chemical approaches to characterize the role of NF-κB in the anti-apoptotic response. MATERIALS AND METHODS Toxoplasma gondii T. gondii tachyzoites (RH) were maintained in the peritoneal cavity of ICR mice (Dae-Han BioLink Co., Eumseong-gun, Chungcheongbuk-do, Korea) for three to four days. Tachyzoites were harvested in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) from the peritoneal cavity and separated from peritoneal cells by repeated low and high speed centrifugation (40 × g for 5 min and 650 × g for 10 min). Treatment of mouse spleen cells with T. gondii infection and actinomycin-D Spleen cells in RPMI 1640 media were harvested from BALB/c mice and pelleted by centrifugation (650 × g for 10 min). Cells were resuspended in lysis buffer (0.83% NH4Cl-0.01M Tris-HCl) in order to remove red blood cells. Mouse spleen cells (5 × 106 cells/well) were cultured in 6-well plates with RPMI 1640 media containing 2 mM L-glutamine, 2 mg/mL NaHCO3, 10% fetal bovine serum (FBS, Cambrex, Walkersville, MD, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 , 5% CO2 atmosphere. Cells were infected with 5 × 107 T. gondii tachyzoites and/or treated with 5 - 10 μg/mL actinomycin-D (AD, Sigma- Aldrich, St. Louis, MO, USA) for 5 h and 18 h, respectively. DNA size analysis by agarose gel electrophoresis Infected and non-infected mouse spleen cells were harvested by centrifugation and resus- pended in NET buffer containing 10% SDS, DNA- free Rnase, and proteinase K. The cells were then centrifuged at 12,000 × g for 5 min at 4 , and DNA was extracted from the cell pellet with phenol, phenol/isoamyl alcohol, and chloroform, followed by precipitation with 100% ethanol and 10 M NH4OAc. The DNA was dissolved in TE buffer and quantified by A260 on a spectrophoto- meter (Perkin Elmer, Wellesley, MA, USA). DNA samples were analyzed on a 2% agarose gel stained with 1 μg/mL ethidium bromide. Flow cytometry Cells were stained with annexin V using the Annexin V/PI kit (R&D systems, Minneapolis, MN, USA), and apoptotic cells identified and quantified by flow cytometry. Briefly, spleen cells (1 × 106) were washed in PBS and incubated with 10 X binding buffer, propidium iodide (PI), annexin V-FITC, and dH2O (total 100 μL) for 15 min at room temperature. The apoptotic cells were analyzed on a fluorescence-activated cell sorter (FACS, Becton Dickinson-FACScalibur, Cockeysville, MD, USA). Reverse transcriptase (RT)-PCR Bcl-2 family messenger RNA was identified by RT-PCR using message specific primers. Mouse spleen cells were incubated with AD and AD/T. gondii for 30 min, 60 min or 120 min, and total cellular RNA extracted using TRIzol (Invitrogen), chloroform, and isopropyl alcohol. Reverse tran- scription was performed with an oligo dT primer, and cDNAs were amplified by PCR using the primers listed in Table 1. PCR products were resolved on a 2% agarose gel stained with 1 μg/ mL ethidium bromide. Western blot analysis of Bcl-2 family proteins and caspases The abundance of Bcl-2 family proteins (Bax, Bcl-2, Bcl-XL), caspase 3, caspase 8, PARP and actin in mouse spleen cells was analyzed by Western blotting. Spleen cells (5 × 105) were re- suspended in lysis buffer (20 mM Tris-HCl, PH Yonsei Med J Vol. 47, No. 6, 2006