foetus. Polystyrene microspheres (Polysciences, USA) with a mean diameter of 0.96 μm were re-suspended in PBS to 1 × 108 particles/ml. These particles were allowed to interact with the parasites in TYM medium without serum with a parasite:latex bead ratio of 1:10 for 45 min at 37 °C. After interaction, cells were

fixed and processed for scanning electron microscopy analysis as outlined below. Quantitative analyses of different shapes of T. mobilensis for adherence with uncoated polystyrene microspheres incubated for 45 min were performed, and two thousand parasites were counted using SEM. Statistical significance of binding was evaluated by a 2-way ANOVA. In all check details cases, a P-value <0.05 was considered significant. Cells were fixed FG-4592 chemical structure in 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.2), post-fixed for 15 min in 1% OsO4, dehydrated in ethanol, critical point dried with CO2 and sputter-coated with gold-palladium. The samples were examined with a JEOL 5800 scanning electron microscope. Cells were fixed in 2.5% (v/v)

glutaraldehyde, post-fixed for 15 min in 1% OsO4, dehydrated in acetone and embedded in Epon. Ultra-thin sections were observed with a JEOL 1210 transmission electron microscope. TEM images were captured using a Megaview G2 digital camera (Olympus; Muster, Germany). The diameter (μm) and area (μm2) of T. mobilensis and T. foetus hydrogenosomes unless were measured using iTEM software (Olympus; Munster, Germany). Approximately 250 hydrogenosomes were measured for each parasite species. Statistical significance was evaluated by a 1-way ANOVA. In all cases, a P-value <0.05 was considered significant. Ethanol preserved cells were harvested by centrifugation at 20,000 × g and washed in TE (10 mM Tris–HCl, pH 8.0; 1 mM EDTA) buffer. The digestions were carried out in lysis buffer (10 mM Tris–HCl, pH 8.0; 5 mM EDTA;

1% SDS) with proteinase K. Genomic DNA was isolated by a standard two-step phenol/chloroform extraction ( Sambrook and Russell, 2001). RNase treatment followed the first phenol/chloroform step. The ITS-1/5.8S/ITS-2 genomic region was amplified with the following primers: NC5 (forward primer 5′-GTA GGT GAA CCT GCG GAA TCA TT-3′) and NC2 (reverse primer 5′-TTA GTT TCT TTT CCT CCG CT-3′) ( Newton et al., 1998). PCR was performed in a total volume of 20 μl using approximately 20 ng of genomic DNA, 0.2 μM of each primer, 0.2 μM of each dNTP, 3 mM MgCl2 and 0.5 U Taq DNA polymerase (Invitrogen; USA) with the following conditions: 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C for 35 cycles. Post-extension at 72 °C was performed for 5 min. For each set of PCR reactions, negative (without DNA) and positive (using DNA extracted from T. foetus) controls were included. PCR products were purified using exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences, USA).