After 48-72 h the parasites were harvested in PBS and centrifuged (200 g for 7-10 min) at room temperature in order to
discard blood cells and cellular debris. The supernatant was collected and then centrifuged again at 1000 g for 10 min. The final pellet was resuspended in DMEM and used in the interaction assays. T. gondii infection during skeletal muscle cell myogenesis Aiming to verify the infectivity of T. gondii in myoblasts and myotubes, we developed the following protocol: 2-day-old cultures were infected with tachyzoite forms (1:1 parasite-host cell ratio) and, after 24 h of interaction, the total number of infected myoblasts and myotubes was quantified independent of the number of internalized parasites. For evaluation of the potential interference of T. gondii in myotube formation, after the initial seeding, cultures were maintained for 48 h in medium without calcium, in order to not stimulate myoblast fusion. buy BGB324 After this time, the cultures, enriched in myoblasts, were infected for 24 h. Cell fusion in the presence or absence of T. gondii was determined by morphological analysis of myoblast alignment and the observation of the percentage of multinucleated cells. The quantitative analysis was based on 3 independent experiments performed in duplicate with at least
200 cells in each selleck products coverslip. Fluorescence analysis of actin microfilaments SkMC 2-day-old cultures were allowed to interact with tachyzoites (1:1 parasite: host cell ratio) for 24 and 48 h at 37°C. Non-infected and infected SkMC were fixed for 5 min at room temperature in 4%
paraformaldehyde (PFA) diluted in PBS. After fixation, the cultures were washed 3 times (10 min each) in the same buffer. Then, the cultures were incubated for 1 h at 37°C with 4 μg/ml phalloidin-rhodamine diluted in PBS. Thereafter, the cultures were washed 3 times (10 min each) in PBS, incubated for 5 min in 0.1 μg/mL DAPI (4′,6-diamidino-2-phenylindole, Sigma Chemical Co.), a DNA stain that enables the visualization of host and parasite nuclei, and washed again in PBS. The coverslips were mounted on slides with a Fenbendazole solution of 2.5% DABCO (1,4-diazabicyclo--octane-triethylenediamine antifading, Sigma Chemical Co.) in PBS containing 50% glycerol, pH 7.2. The samples were examined in a confocal laser scanning microscope (CLSM Axiovert 510, META, Zeiss, Germany) from the Confocal Microscopy Plataform/PDTIS/Fiocruz, using a 543 helium laser (LP560 filter) and 405 Diiod laser (LP 420 filter). Immunofluorescence analysis of total cadherin protein distribution in SkMC myogenesis during infection with T. gondii Immunofluorescence assays were performed using specific monoclonal antibodies for pan-cadherin (Sigma Chemical Co. C3678). Briefly, tachyzoite forms were allowed to interact with 2-day-old SkMC in the ratio of 1:1.