, 1995). Surprisingly, thus far a bona fide TGF-β from TCT has never been characterized. T. cruzi penetration into host cells also leads to intracellular Ca2+ mobilization, both in trypanosome and target cells ( Yoshida, 2006). Ca2+transients are necessary selleck for rapid rearrangements in the host cell cytoskeleton and recruitment of host cell lysosomes ( Rodríguez et al., 1995 and Rodríguez et al., 1996). Experimentally buffering host cell intracellular free Ca2+ or depleting intracellular Ca2+ stores abrogated parasite invasion ( Tardieux et al.,

1994 and Rodríguez et al., 1995). The objective of the present study was to investigate whether T. theileri possesses intracellular amastigote stages in in vitro cells. If it can be proven, thereby the related molecular events of parasite-host cell interactions can be characterized and will also become the corroborating evidence for T. theileri cell invasion. A T. theileri TWTth1 strain

was obtained from supernatants of infected BHK cells, as described in our previous study ( Lee et al., 2010). Cultured trypanosomas were cryopreserved and the parasites were controlled under low culture passage, with no more than six passages through the entire experimental procedure. Amastigotes were generated in culture by incubating freshly harvested trypomastigotes in liver infusion tryptose medium containing 10% FBS (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2 for 72 h. BHK (baby hamster kidney cell), SVEC4-10 (mouse lymph node endothelial cell), H9c2(2-1) (rat heart myoblast) and RAW 264.7 (mouse monocyte/macrophage cell) PFT�� supplier cell lines were obtained from the Bioresource Collection and Research Centre in the Food Industry Research and Development Institute (BCRC, FIRDI), Hsinchu, Taiwan, and were cultured according to the manufacturer’s instructions. Tissue culture trypomastigotes (TCT) were obtained from the supernatants of infected BHK cells cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% FBS. A pure suspension of TCT from a culture of infected BHK cells was obtained by collection and centrifugation of the medium at 3000 × g for 15 min. Highly motile trypomastigotes were

then recovered following swimming up to the supernatant fraction for 3 h in DMEM at 37 °C in an atmosphere of 5% CO2. To ensure Isotretinoin infectivity, trypanosome-infected culture cells were prewashed with phosphate-buffered saline (PBS) and fixed in methanol followed by Giemsa staining. Transmission and scanning electron microscopy (TEM, SEM) was used for examination, as previously described (Lee et al., 2010). For T. theileri attachment and invasion assays, the standardized procedure ( Lima and Villalta, 1989) was modified in this study. BHK, H9c2, SVEC and RAW 264.7 cell lines (BCRC, FIRDI) were grown at 37 °C in 24-well plates containing 1 × 105 cells in DMEM with 10% FBS in an atmosphere of 5% CO2. To measure attachment, cells were infected with TCT at a proportion of 20:1 parasites/cell.