2). At 3, 8 and 12 hours of infection, the microorganisms were detected mostly surrounding the perinuclear regions (check details Figure 1.3 and 1.4). The studied microorganisms showed

no differences in their distribution when adhered to or inside the cytoplasm after 12 hours of infection. Ureaplasmal infection produced no GSK2126458 cytopathic effects in Hep-2 cells in the studied period. Figure 1 Infection of U. diversum in HEp-2 cells. LSCM optical sections showing internalization of U. diversum in HEp-2 cells after 1 minute (1), 30 minutes (2), 3 hours (3) and 12 hours (4) post-infection. Ureaplasmas were labeled with Vibrant Dil (in red, A), HEp-2 actin filaments stained with phalloidin-FITC (in green, B) and Hep-2 nuclei stained with TO-PRO-3 (in blue, C). In D, merging images A, B, and C. One minute after infection, ureaplasmas were observed inside HEp-2 cells, and after 30 minutes the presence of ureaplasmas inside cells increased. After 3, 8 and 12 hours of infection, ureaplasmas were observed throughout cells cytoplasm. Disposal of U. diversum in the infected HEp-2 cells Figure 2 shows disposition of ureaplasma in the studied infection. In figure 2A, optical slices from basal to

apical regions of cells, including sections with the nucleus in the plane of the focus were also obtained. The ureaplasmas were detected in different sections of the Hep-2 cell cytoplasm but not inside the nucleus. The orthogonal sections after 3 hours of infection showed a red fluorescence from apical to basolateral regions and throughout the cytoplasm and perinuclear selleckchem spaces. In figure 2B, images of the tri-dimensional distribution filipin of Hep-2 cells three hours after infection were focused. As shown in figure 2A, red fluorescence was detected throughout the cytoplasm and perinuclear spaces. Figure 2 Distribution of U. diversum in infected HEp-2 cells. LSCM images showing the internalization of U. diversum in HEp-2 cells.

Ureaplasmas stained by Dil (in red), actin filaments stained by phalloidin-FITC (green) and cells nuclei stained by TO-PRO-3 (in blue). A and B: Z-series of optical slices (A) and orthogonal projection (B) showing the presence and distribution of ureaplasmas inside HEp-2 cell. C: Image and graphic representation of HEp-2 cells after 12 hours post-infection. The arrow in confocal image indicates the cell in which the ureaplasma (in red) and actin (in green) was analyzed, and the detection of actin and ureaplasmas throughout this cell is represented in the graphic. D: Infected HEp-2 cells submitted to immunofluorescence with anti-lamin antibody (in green), showing ureaplasmas (in red) in the perinuclear region, but not inside the cell nuclei. All the images show ureaplasmas distributed throughout the HEp-2 cytoplasms, and concentrated in the perinuclear region, surrounding the nuclei. Figure 2C is the graphic representation obtained with the software Imaris 3.1.