Detection of intracellular parasites within the ring mask.

<p>Macrophages were cultured in 96-well cultures in the presence (plus <i>L</i>. <i>major</i>, right row) or absence of <i>L</i>. <i>major</i> parasites (w/o <i>L</i>. <i>major</i>, left row). After 72h a DAPI staining was performed to visualize nuclei of the mammalian cells and the DNA rich areas of the parasites simultaneously. Single channel greyscale pictures were processed by TissueQuest software. The ring masks were created as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.g001" target="_blank">Fig 1</a> and material and methods. (A) The ring mask (highlighted in blue) visualizes the cytoplasmatic area of a representative macrophage that was cultured in the absence of parasites. (B) One representative macrophage infected with <i>L</i>. <i>major</i> parasites is shown including the ring mask which is highlighted in blue. Visually determined DAPI positive signals are marked with yellow arrows. For the detection of parasites within the ring masks a virtual channel of the parasite-associated fluorochrome (in our case DAPI) has to be created (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.s002" target="_blank">S2C Fig</a>). The following instrument settings were used: Use ring mask: yes, interior radius: -0,31 μm, exterior radius: 12,74 μm, use identification mask: no, use nucleus mask: no, background threshold: 5. (C) One algorithm developed for the detection of weak signals recognizes false positive signals (yellow squares) within the ring mask (the cell shown in A is highlighted in blue) of macrophages that are not infected. (D) False positive signals (yellow squares) are created. The representative (highlighted in blue) macrophage harbors more than 30 parasites, whereas no more than 10 can be visually determined (see B). (E) The algorithm that was developed to detect weak signals recognizes no false positive signals in the ring mask of macrophages that are not infected. (F) All parasites within infected macrophages are recognized (see yellow squares and yellow arrows in (B)). Representative pictures out of 3 experiments are shown. Bar = 10 μm.</p

Phenotypical analysis of macrophages harboring different numbers of parasites.

<p>Macrophages were cultured in 96-well plates in the presence of <i>L</i>. <i>major</i> parasites (parasite/macrophage ratio 5:1). After 72 hours the cells were stained with F4/80^Cy5, CD11b^biotinylated + Streptavidin^AF546. Macrophage nuclei and parasite DNA rich areas were stained with DAPI. (A) The TissueQuest-based analysis of cells and intracellular parasites are presented. A histogram plot depicts the number of parasites within the macrophages (x-axis) and the number of macrophages harboring the indicated (see x-axis) number of <i>Leishmania</i> parasites. Cells within the highlighted gates were further analyzed regarding the expression of CD11b and F4/80. (B) The mean intensities of CD11b or F4/80 are plotted representing macrophages harboring no (#0), one (#1), six (#6) or ten (#10) parasites. Statistical analyses were performed with GraphPad Prism using the nonparametic Mann-Whitney test (p**<0,01, p*<0,05; red horizontal line represents the median). Every single dot represents one individual analyzed nucleated cell. One representative set of data out of two experiments is shown.</p

Nuclei detection and ring mask calculation by TissueQuest.

<p>Single channel greyscale pictures were processed by TissueQuest software. (A) The nuclei detection was performed according the following parameters (nuclei size: 10, remove small objects: 1, remove weakly stained objects: 1, automated background: no, automated threshold: 5, virtual channel: no, post processing order: remove, merge and Remove labels i) smaller then: 30 μm, larger then: 100 μm, weaker then: 137, stronger then: do not use; use merging rules: no). The detected nuclei are automatically surrounded by a red line. (B) The ring masks were created according the following parameters (interior radius: -0,31 μm, exterior radius; 12,74 μm, use identification cell mask: no, use nucleus mask: no, background threshold: 5; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.s001" target="_blank">S1 Fig</a>). The white insert highlights one infected macrophage. The white arrows depict some of the parasites within the ring mask. (C) The area within the ring mask is highlighted in yellow and represents the region of interest in which the screening of parasites can be performed automatically. Representative pictures out of 3 experiments are shown. Bar = 10μm.</p

Assessment of parasite load <i>in situ</i>.

<p>The skin-draining lymph nodes of infected BALB/c mice were removed 20 days after infection. Cryosections were stained with DAPI and analyzed by fluoresence microscopy. (A) One representative region of the infected lymph node is shown. The gray scale image depicts DAPI⁺ host cell nuclei and parasite DNA. (B) An overlay of the detection of nuclei (green), cytoplasm (orange), and parasites (magenta) is shown. (C) Backward gating visualizes cells harboring 3 and (D) 5–10 parasites (highlighted in magenta). E) The graph represents the parasite load (y-axis) within infected cells. The horizontal line represents the median and the arrow bars display the interquartile range. Each dot represents a host cell harboring the indicated (y-axis) amount of parasites.</p

Baseline study characteristics.

<p>Data are presented as mean ± SEM, ND not determined, LFD low fat diet, HFD high fat diet, WT wildtype, PC Phosphatidylcholine, BW body weight.</p

Absence of CD40L does not ameliorate insulin sensitivity in diet-induced obesity.

<p>Plasma glucose (A) and insulin levels (B) were determined in animals fasted overnight (A) and for 6 hours (B) overnight. Insulin tolerance (ITT) and glucose tolerance testing (GTT) were performed after intraperitoneal insulin (0.5 U/kg lean body mass) or glucose (1 g/kg lean body mass) injection (C–F). Inlays represent area under the curve calculation (AUC) of the indicated glucose curve.</p