Rationale behind the presented approach.

<p>The figure shows the relation of the size of T cells to the height of the used tissue sections (2 µm). The sections are so thin, that there is only minimal overlap between the individual cells of a conglomerate. This allows for calculating the number of cells in a conglomerate by its total area.</p

Conglomerates and single cells.

<p>Liver metastasis of colorectal cancer with strong T cell infiltrate (CD3 staining: dark red with hematoxylin counterstaining, A: overview, digital magnification 10×, B: conglomerate (magnification 40×), C: single cells, (magnification 40×).</p

Cell counts for 30 different fields with one ore more conglomerates, each evaluated by two observers (“Manual1” and “Manual2”) and the here presented algorithm (“Automated”).

<p>Note the up to 41% variation between the observers at high cell counts (1000-1.500). For quantitative data comparison see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007847#pone-0007847-t001" target="_blank">table 1</a>.</p

Exemplary workflow for the described algorithm.

<p>Stained immune cells are either counted individually (where possible) or the number of cells is estimated by the conglomerate surface. Both results are added.</p

Repeated quantification (six times) of a large conglomerate by one observer.

<p>Triangles show single values for each repetition and thin vertical lines indicate range, thick horizontal line indicates average value.</p

Cell area sizes of T cells (CD3+) measured in histological sections of different tissues with 2 µm thickness.

<p>Cell area sizes of T cells (CD3+) measured in histological sections of different tissues with 2 µm thickness.</p

Impact of cell counts from conglomerates on total cell counts in 10 individual 1 mm² fields from sections of 10 different patients.

<p>Omitting conglomerates in the quantification would substantially distort the total cell counts.</p

Cell counts of T cells (CD3+) in thirty different fields of 1 mm² size.

<p>Fields with maximum differences between manual cell counts are highlighted.</p><p>Abs. Diff. Man. = Absolute Difference between Manual1 and Manual2, % Diff. Man. = Percentage Difference between Manual1 and Manual2.</p

Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration

<div><p>Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes <i>in vitro</i>; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. <i>In vitro</i>, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.</p> </div

Increased Stathmin levels correlate with keratinocyte proliferating in an OTC wound healing model.

<p>(A) Schematic display of sample segmentation for automated analysis of OTC wound specimens. Segmentation in 10 areas was defined using HE-staining, while quantitative evaluation was performed using consecutive sections after Ki67/Stathmin double staining. Opposite areas were pooled (A/A’, B/B’, C/C’, D/D’, E/E’) to increase the cell number suitable for statistical analysis. (B) Ki67 and Stathmin double staining of OTC wound healing sections. Exemplary wound edges were shown for 0 (immediately after wounding), 1, 4, and 6 days after punching. (C) Percentage of Stathmin (left) and Ki67 (right) positive cells in different regions of the OTC specimens at indicated time-points. Please note that the regions E/E' (for day 1) and D/D’; E/E’ (for day 0) are not detectable due to incomplete wound closure at early time points. (D) Exemplarily, Ki67/Stathmin double staining is shown. Results of automated quantitative analysis of Ki67 and Stathmin positivity in keratinocytes of the basal epidermal layer are depicted. Black arrows: Ki67-negative/Stathmin-positive cells; White arrows: Ki67/Stathmin-positive cells. Dashed line in bar graph indicates the mathematical regression for the whole experimental time-course. Two independent time-courses were analyzed showing similar results.</p