A and B: Forearm skin biopsy staining for SDF-1in a diabetic patient (Figure 4A) and a healthy control subject (Figure 4B), (frozen sections, x100).

<div><p>SDF-1 was expressed by stromal cells (black arrows) and endothelial cells (red arrows) and the staining pattern was mostly cytoplasmic and occasionally nuclear in cases of increased expression. The number of stained stromal cells and the intensity of staining were increased in in diabetic patients while no difference was found in the number of stained endothelial cells.</p> <p><i>C</i> and <i>D</i>: Foot skin staining for CXCR4 in a diabetic patient (Figure 4C) and a healthy control subject (Figure 4D) (frozen sections, x200). CXCR4 was expressed by stromal cells (black arrows), endothelial cells (red arrows) and epithelial cells (blue arrows) and the staining pattern was mostly membranar and cytoplasmic. The intensity of staining was higher in in the diabetic group (p<0.05) but no differences were observed between the two groups in the number of positive stromal and endothelial cells (p=NS).</p></div

NanoFCM allows identification of beads and liposomes down to 100 nm.

<p>Separation of a mixture containing 200 and 500 nm latex beads by LSRII (A), and NanoView (B) instruments show more distinct separation with the NanoView Instrument. The NanoView is capable of separating a mixture of 100–500 nm beads into distinct populations (C) and can detect 100 nm liposomes (D). The gating strategy for these experiments to determine instrument and background noise are described in the methods section.</p

EVs detected in plasma from a healthy donor.

<p>Plasma from 5 mL of blood was collected, centrifuged to remove cellular debris (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144678#sec002" target="_blank">methods</a>), and imaged following a series of dilutions in PBS (A) to test for ‘swarming’. An EV population (based on positioning of 100 nm liposomes and size distribution of plasma samples) from the plasma of a healthy donor was sorted (gate R2; B) and imaged using atomic force microscopy (C). The size distribution of sorted EVs was analyzed by qNano and is represented in D. The gating strategy for these experiments is detailed in the methods section.</p

An example of flow cytometric analysis of human peripheral blood sorted on CD45^dim cells in a patient with DFU (A) and a healthy control subject (B).

<p>The triple positive phenotype (CD34⁺/KDR⁺/CD133⁺) was determined by gating the CD133⁺ cells on the CD34⁺/KDR⁺. 1.000.000 events per sample were acquired and the counts for each phenotype are shown in the picture. Smaller counts in all phenotypew were observed in the diabetic patient (A) when compared to the healthy subject (B) in the double and triple measurements. </p

Changes in EPCs measurements during the four study visits between the patients who did not heal their ulcers (NH) and those who did (H).

<p>There were no differences in all EPC measurements at baseline but patients who healed their ulcers had lower CD34⁺KDR⁺ counts at visits 3 and 4 and CD34⁺CD133⁺ at visit 4. Data are presented as the median and interquartile range box.</p

Changes in CRP (3a), IL-1a (3B) and GM-CSF (3C) during the four study visits between the patients who did not heal their ulcers (NH) and those who did (H).

<p>Data are presented as the median and interquartile range box. Patients who healed their ulcers had lower serum CRP and GM-CSF at visit 1 and lower IL-1α at visits 1 and 4.</p