Phagocytosis of fluorescent latex beads by GM-Mφ and M-Mφ generated from monocytes isolated by negative (neg.) or positive (pos.) selection as measured by flow cytometry.

<p>Histogram plots show macrophages incubated with latex beads (hatched area) and cells alone (black lines) as control. Percentage of macrophages which are positve for latex beads are indicated in each histogram plot. Results from one representative donor are shown and similar results were obtained with cells of at least three different donors.</p

Surface expression of CD14, CD163 and CD206 on untreated THP-1 cells (mock) or after differentiation into macrophage-like cells by stimulation with PMA alone (PMA) or in combination with 50 ng ml⁻¹ M-CSF (M+PMA) or GM-CSF (GM+PMA).

<p>All experiments were performed in triplicates (n = 3). Histogram plots show surface marker staining as hatched areas and isotype controls as black lines.</p

Fluorescence microscopy of GM-Mφ (GM) and M-Mφ (M) generated from monocytes isolated by negative (neg.) or positive (pos.) selection and infected with <i>Lm</i> at an MOI of 10 (A).

<p>Nuclei of macrophages are stained with DAPI (blue) and <i>Lm</i> was stained with a specific antibody (red). Scale bar is 10 µm. Infection of GM-Mφ (-GM or +GM, black bars) and M-Mφ (-M or +M, white bars) with <i>Lm</i> was determined at different multiplicities of infection (MOI) and fluorescence microscopy images were analysed either for the percentage of infected cells (B) or the number of bacteria per infected cell (C). For each MOI and macrophage phenotype, infected cells and <i>Lm</i> per infected cell were counted in random microscopic fields of view of three donors. For each donor, three independent fields of view with at least 100 cells were analysed. Statistical analysis was performed on the means of different donors by pairwise comparison of GM-Mφ vs. M-Mφ at the different MOIs using Student's <i>t</i>-test (n.s.: not significant).</p

Phagocytosis of <i>Lm</i> by GM-Mφ (GM, black bars) or M-Mφ (M, white bars) generated from monocytes isolated by negative (A) or positive (B) selection or THP-1 macrophages (C) at different multiplicities of infection (MOI).

<p>Phagocytosis is measured as colony forming units (CFU) per well. Results from one representative donor (A and B) or experiment (C) measured in triplicates are shown and similar results were obtained with cells of at least three different donors or in three independent experiments. Statistical analysis was performed by pairwise comparison of GM-Mφ vs. M-Mφ at the different MOIs using Student's <i>t</i>-test (n.s.: not significant).</p

Phagocytosis of <i>Lactococcus lactis</i> (A), <i>Escherichia coli</i> (B), <i>Leishmania major</i> (C) and HCMV (D) by GM-Mφ (GM, black bars) and M-Mφ (M, white bars) generated from monocytes isolated by negative (neg.) or positive (pos.) selection.

<p>Cells were infected with all microorganisms at an MOI of 1. Phagocytosis was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066898#s2" target="_blank">Materials and Methods</a> expressed as CFU/well or infection rate (%). Results from one representative donor measured in triplicate are shown and similar results were obtained with cells of at least three different donors. Statistical analysis was performed by pairwise comparison of GM-Mφ vs. M-Mφ using Student's <i>t</i>-test.</p

Viability of GM-Mφ and M-Mφ generated from monocytes isolated by positive (+GM or +M) or negative (-GM or -M) selection (A) or PMA-stimulated THP-1 macrophages (B) infected with <i>Lm</i> at different multiplicities of infection (MOI).

<p>Results from one representative donor (A) or experiment (B) measured in triplicate are shown and similar results were obtained with cells of at least three different donors or in three independent experiments. Statistical analysis was performed by pairwise comparison to the respective uninfected controls using Student's <i>t</i>-test.</p

Phagocytosis of <i>Lm</i> by GM-Mφ (GM, black bars) and M-Mφ (M, white bars) generated from monocytes isolated by negative (neg.) or positive (pos.) selection as well as negatively isolated macrophages, which were additionally incubated with the CD14 microbeads (neg.+MB; A) or the α-CD14 antibody (neg. +AB; B).

<p>Cells were infected with <i>Lm</i> at an MOI of 1. Results from one representative donor measured in triplicate are shown and similar results were obtained with cells of at least three different donors. Statistical analysis was performed by pairwise comparison of GM-Mφ vs. M-Mφ using Student's <i>t</i>-test (n.s.: not significant).</p

Surface expression of CD14, CD163 and CD206 on GM-Mφ (A) and M-Mφ (B) derived from monocytes isolated by negative (neg.) or positive (pos.) selection.

<p>Histogram plots show surface marker staining as hatched areas and isotype controls as black lines. Results from one representative donor are shown and similar results were obtained for each isolation and phenotype with cells of at least three donors. (C) Fluorescence intensity of surface marker staining on primary macrophages. Values are mean fluorescence intensity (MFI) ± standard deviation obtained with cells of three different donors.</p