After incubation, Hoechst staining to label the nucleus and kinetoplasts was performed for 20 min. In addition, positive autophagy control was induced by washing grown cells three times with PBS and incubating them with starvation media (DMEM without FBS) at 37 °C for 24 h. During autophagosome formation, LC3-GFP is processed and recruited to the autophagosome membrane, where it can be imaged as cytoplasmic puncta by confocal fluorescence microscopy.

Changes in Ca2+ concentration were detected with the fluorescence probe Fluo-2/AM based on a modified version of a previously described protocol (Moreno et al., 1994). Briefly, H9c2 cells were plated for 48 h before the assay at 5 × 103 per dish, on a 35 mm glass bottom, and loaded with 5 mM fluo-2/AM (Molecular Probes Inc.) for 60 min at 37 °C in the dark. After loading, MDV3100 the dishes were washed in Hepes-buffered Ringer’s solution. The cells on cover slips were then transferred to a microchamber on the stage of an inverted Olympus microscope, and viewed under bright light and UV illumination using a 40× oil immersion

fluorescence objective. Selleckchem Alectinib Ca2+ concentration was monitored for 1 min at the basal level. Fluorescence images were collected with a video camera (CCD-C72; Dage/MTI, Michigan city, IN) through a 340 nm objective and recorded on an optical memory disk (TQ-3031F; Panasonic, Secaucus, NJ), at time-lapse intervals of 1 s using a computer-controlled shutter system (Photon Technology International; PTi Corp., Birmingham, NJ). Ca2+ was monitored by alternating excitation wavelengths of between 340 nm and 380 nm and emission at 510 nm with a Delta Scan System from (Photon Technology International; Princeton, NJ), and parasites were added to the cells at 50 s after initiation of the time-lapse recording. All the experiments were carried out at 30 °C. All data are expressed as mean ± SD. Descriptive statistics were first used for analysis of normality. A Bonferroni t-test or a Mann–Whitney Rank Sum Test was used depending on the normality of data. The mean values of two groups were considered significantly

different if *P < 0.05, **P < 0.01, or ***P < 0.001. To ensure the initial step of invasion, parasite-infected culture cells were observed under light microscopy (Giemsa staining), TEM and SEM (Fig. 1). Parasites were strongly bound to the cell surface and could not be dislodged even with extensive washings with PBS. Because TCT is a pleomorphic population, different forms of trypomastigotes were found to be attached to the cells (Fig. 1A and B). In TEM images, T. theileri was attached to BHK cells ( Fig. 1C). After infection of H9c2 cells, Giemsa staining showed that parasites had replicated inside the PV ( Fig. 2A). The self-prepared mouse polyclonal antibody for T. theileri was specifically bound to TWTth1 including different forms ( Fig. 2B, bottom left panel), but not to host cells ( Fig. 2B, upper left panel, arrow). T. theileri were multiplied in the cytosol of H9c2 cells ( Fig.