Phagocytosis and Microbicidal activity were evaluated by the acridine orange method described by Bellinati-Pires et al. Equal volumes of bacteria and cell suspensions were mixed and incubated at 37Â°C for 30 min under continuous shaking. Phagocytosis was stopped by incubation in ice. To eliminate extracellular bacteria, the suspensions were centrifuged twice (160 Ã g, 10 min, 4Â°C). Cells were resuspended in serum-free 199 medium and centrifuged. The supernatant was discarded and the sediment dyed with 200Î¼L of acridine orange (Sigma, ST Loius, USA; 14.4g/L) for 1 min. The sediment was resuspended in cold culture 199 medium, washed twice and observed under immunofluorescence microscope at 400X and 1000X magnification.
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Last date updated on September, 2014