Time costs of the surveys (^a three students working simultaneously).

<p>Time costs of the surveys (^a three students working simultaneously).</p

Responses to nonscaled^‡ and Likert-scaled* questions for the three survey methods (E₁, H₁, and P₁).

<p>Responses to nonscaled^‡ and Likert-scaled* questions for the three survey methods (E₁, H₁, and P₁).</p

Number of participants and effective survey responses; ^aoccasionally, computers were professionally used by several employees.

<p>Number of participants and effective survey responses; ^aoccasionally, computers were professionally used by several employees.</p

Estimated costs for electronic and paper-based questionnaires with 10, 20, 30, or 42 questions and different cohort sizes.

<p>Costs are calculated with student working hours ($17.4/h), except for the implementation of the questionnaire in an Internet program ($77.8/h, 8 h professional work), and are based on our results according to the different cohorts.</p><p>Estimated costs for electronic and paper-based questionnaires with 10, 20, 30, or 42 questions and different cohort sizes.</p

Financial costs of our surveys and estimated costs based on ^a77.8/h,8hprofessionalworktoestablishadigitalquestionnaire,^b77.8/h, 8 h professional work to establish a digital questionnaire, ^b0.05/copy-page, $17.4/h, students working hours (q = questions, p = participants).

<p>Financial costs of our surveys and estimated costs based on ^a$77.8/h, 8 h professional work to establish a digital questionnaire, ^b$0.05/copy-page, $17.4/h, students working hours (q = questions, p = participants).</p

Estimated costs for electronic and paper-based surveys with different cohort sizes and numbers of questions based on the surveys E₁ (centralized e-mails), H₁ (homepage link), and P₁ (paper-based questionnaires).

<p>Estimated costs for electronic and paper-based surveys with different cohort sizes and numbers of questions based on the surveys E₁ (centralized e-mails), H₁ (homepage link), and P₁ (paper-based questionnaires).</p

CCR2-deficiency temporarily reduced CNV areas in choroidal flatmounts.

<p>(A) Fluorescence microscopy of Isolectin B4-stained choroidal flatmounts of eyes from CX₃CR1-GFP reporter mice showing the endothelium in neovascular areas with GFP⁺ phagocytes (single spot; scale bar = 100 μm). (B) Isolectin B4 (IB4)-positive CNV areas from CX₃CR1 reporter mice lacking (black bar) or expressing CCR2 (black bar) after 2 (2w) or 3 weeks (3w) following laser treatment. Bar graphs show means ± SD. A total of 3–5 mice were used per group 2 independent experiments. CNV areas were analyzed by the two-tailed nonparametric Mann-Whitney-U-Test. The statistical significance was analyzed as indicated (*p<0.05, n.s.  =  not significant).</p

CD11b⁺ mononuclear phagocytes, CD11c⁺ dendritic cells, endothelial cells and retinal vimentin⁺ cells contain VEGF.

<p>(A) VEGF⁺ cells were analyzed for CD11b and CD11c expression. CD11b⁺ CD11c^– phagocytes and CD11b^– CD11c⁺ dendritic cells (DCs) are shown in the choroid and the retina of untreated eyes (left) and 3 days after laser injury (right). (B) The VEGF⁺CD11b⁺ phagocytes were subclassified by analyzing their CD45 and CX₃CR1 expression at day 3. (C) VEGF⁺CD11b⁺ cells in the lasered choroid were further characterized by analyzing F4/80 expression (bottom), which is present on MP but absent on neutrophils. (D) VEGF⁺ endothelial cells (ECs) were identified by CD31 staining in eyes from healthy mice (left) and after laser at day 3 (right) in the choroid (top) and the retina (bottom). (E) Retinal VEGF⁺ cells were identified as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094313#pone-0094313-g001" target="_blank">Figure 1</a> and displayed (top). VEGF⁺ cells were stained positive for vimentin (black line in Histogram) versus the control (grey tinted). (F) Gating strategy to exclude CD31⁺ ECs (1) and CD11b⁺ and CD11c⁺ phagocytes (2) from the vim⁺ cell subset of astrocytes and Müller cells (3). This subset was analyzed for VEGF expression compared to the isotype control. (G) RPE cells were stained for the marker RPE65 (right) versus control (left) in the choroid (top) and the retina (bottom). Percentages (A–D) are means of the respective group. Results are representative for two independent experiments. A total of 3–5 mice per group were analyzed in each experiment.</p

Flow cytometric analysis of VEGF-containing eye cells in the choroid and the retina.

<p>VEGF-containing eye cells from the choroid and the retina of untreated (middle) and laser-treated (right) mice are shown. Specificity was verified by an isotype control (left). Cells positive for VEGF were plotted versus an empty fluorescence channel. Percentages are means of the respective group. Dot-plots display representative data from one of two independent experiments. A total of 4–5 mice were used per group in each of these experiments</p

Higher VEGF content in CD11b⁺ phagocytes but neither in other phagocytes nor non-immune cells after laser injury.

<p>(A) VEGF⁺ CD11b⁻ CD11c⁺ DCs were enumerated in the choroid (top) and the retina (bottom). (B) VEGF content of these DCs without and after laser treatment (histogram). (C) Number of VEGF⁺ astrocytes and Müller cells (top) and their content of VEGF (histograms). CD31⁺ ECs and CD11b⁺ CD11c⁺ phagocytes were excluded as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094313#pone-0094313-g002" target="_blank">Fig. 2F</a>). (D) Analysis of cell numbers for VEGF⁺ ECs and CD11b⁺ phagocytes of the choroid and the retina in untreated eyes and 3 and 6 days after laser injury. (E) VEGF-expression of CD11b⁺ phagocytes and ECs without and after laser treatment in the choroid and the retina. Histograms show VEGF content for cell subsets of untreated eyes (small black line) and 3 days after laser injury (thick black line). The isotype control for the respective cell type in lasered eyes (grey tinted) depicts the background fluorescence. Bar graphs show means ± SD. Comparisons of cell numbers (D) were performed using one-way analysis of variances (one-way ANOVA) in combination with Bonferroni multiple-comparison post-test. The statistical significance was analyzed as indicated (* <0.05, ** <0.01, *** <0.001). A total of 3–5 mice per group were analyzed in 2 independent experiments.</p