Under these conditions the parasite attaches to the host cells and minimal internalization occurs. For invasion assays, 3 h incubation at 37 °C was followed by re-incubation in fresh DMEM
with 2% FBS for an additional mTOR inhibitor 72 h to allow the differentiation of internalized parasites into amastigote forms, which are more easily quantified. Cells were immediately fixed with 4% paraformaldehyde (PFA) in PBS and stained with Giemsa. Interaction rates were determined by manual counting in a total of random 100 cells. The total number of parasites attached TCT per 100 cells and the percentage of cells containing attached parasites were calculated. In addition, intracellular parasites were counted to calculate the percentage of cell invasion. After aldehyde fixation, cells were washed with PBS and then permeabilized with PGN solution (PBS, 0.15% gelatin, 0.1% sodium azide containing 0.1% saponin) for 15 min. Samples then underwent GM1 labeling by incubation with a 1 μg/ml cold solution of CTX-B – Alexa Fluor® 488 (Molecular Probes) for 30 min. Chamber slides were mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) and images were acquired on a confocal fluorescence microscope (Fernandes
et al., 2007). PCR amplifications of 477-bp DNA fragments, using oligonucleotide primers DTO154 and DTO155, corresponding to partial catalytic domains of CATL (cdCATL) enzymes were performed, as described previously (Cortez check details et al., 2009). The reactions were performed for 35 cycles at 94 °C (1 min), 56 °C (1 min), and 72 °C (1 min), followed by a final extension of 10 min at 72 °C. The sequence was confirmed by BLAST searches against the GenBank database at the National Center for Biotechnology Information, USA (http://blast.ncbi.nlm.nih.gov/Blast.cgi). For preparation of parasite soluble extract, 3-mercaptopyruvate sulfurtransferase three-day-old cultured pure TCT were harvested by centrifugation (3000 × g, 10 min, 4 °C) in order to clean any residual
from culture medium components. The resulting pellets were sonicated in sterile PBS on ice with a microtip for two 15-s bursts at a setting of 2.5 (Sonics Vibra-Cell VCX 750). Unbroken cells and nuclei were removed by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was then collected, aliquoted, and stored at −80 °C ( Burleigh et al., 1997). The samples were diluted 1:10 in zymography sample buffer. The protein content of supernatants was determined by preparing bovine serum albumin (BSA) solution as the standard curve. Twenty μg of protein from each sample was run on 10% SDS-PAGE containing gelatin (1.0 mg/ml) without previous heating or reduction and electrophoresis was carried out at 4 °C at a constant voltage of 90 V. After electrophoresis, the gels were washed twice with 2.5% Triton X-100 (Sigma, USA) followed by overnight incubation at 37 °C with zymography Ca2+ containing development buffer, pH 7.0.