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Fig. 2. Infection of dendritic cells (DCs) with different developmental stages of L. amazonensis. Fourteen day-old DCs were co-incubated for 21 hours with amastigotes (Am) from different origins (nude Swiss or BALB/c mice) or with either stationary phase promastigotes (SPm) or metacyclic Pm (MPm) at a 4:1 parasite to DC ratio. MPm were purified by centrifugation on Ficoll gradients (MPm Ficoll) or by negative selection using the mAb 3A1 (MPm 3A1). Before contact with DCs, some Am isolated from nude mice and MPm Ficoll were opsonized with Abs by incubation with an Am- or a Pm-specific immune serum (IS), respectively. After staining of the fixed and permeabilised cells with the Am-specific mAb 2A3-26 and an appropriate fluorochrome conjugate, the percentages of infected DCs were determined either by (A) fluorescence microscopy (500 cells counted) or by (B) flow cytometry (25,000 cells counted). (A) Results are expressed as the means + 1 s.d. of two or three different experiments. (B) Representative dot plot of an overall Leishmania-infected DC population after overnight exposure to MPm Ficoll. Almost all cells express the CD11c molecule (y-axis) and the infected cells express the epitope recognised by the 2A3-26 mAb (x-axis).
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