3-Dimensional images of cells in rhodamine-labeled vesicles of chick embryo CAM.

<p>Orthologous projections of z-stacked photomicrographs of the CAM at 200× magnification. Crosshairs indicate cell of interest. <b>A.</b> B16F1 melanoma cells primarily embolized in the overlying capillary plexus (arrowhead) and at the ends of tapering arterioles. <b>B</b>. An hMSC, retaining its shape, adhered in a large vessel (dashed lines) lying beneath the capillary plexus.</p

Distribution of hMSC to arteries/arterioles, veins and capillaries/end arterioles in the CAM.

<p><b>A.</b> Distribution of hMSC compared to lymphocytes and effects of pre-treatment with anti-SLeX and/or anti-α4 integrin (n = 5). <b>B</b>. Distribution in arteries of hMSC from 5 preparations from 5 different donors of marrow repeated 5 times.</p

Blocking paracrine signaling by TNF across all concentrations and preparations.

<p>The average time course for N number of active cells is plotted for cells stimulated in the absence (blue trace, top value of n) and presence (orange trace, bottom value) of sTNFRII, which competes to bind TNF. Concentrations are indicated at left, and the preparation at top.</p

Comparison of single-cell NF-κB activation dynamics for three different LPS preparations.

<p>Intensity represents the relative nuclear localization of p65-dsRed fusion protein, calculated as mean nuclear intensity divided by initial mean cytoplasmic intensity. The concentration for all three preparations was 0.5 µg/mL. The black line shows the average time course for all cells; the light blue traces are ten randomly selected individual cells. The number of active cells (N), as well as the maximum peak amplitude (Peak Amp), and time elapsed until the maximum amplitude is reached (Time to Peak) are also shown. The maximum intensity is indicated by a dot and the two dashed lines indicate how Peak Amp and Time to Peak are determined. The duration of the first peak (Peak Width) is also shown. This value is determined by drawing a horizontal line at the intensity that is halfway between the minimum and maximum peak value. The region above the line and shaded in green denotes the time during which the p65-dsRed nuclear intensity is more than half of the maximum p65-dsRed nuclear intensity. Below each plot, corresponding representative microscope images are shown for the first 200 minutes after stimulation, as labeled.</p

Real time assay of cells in vessels of the chick embryo CAM.

<p><b>A.</b> Schematic for injecting cells or beads into a large vein of the CAM and capturing images for 3 to 10 minutes at either 40× or 100× magnification. <b>B. (upper panel).</b> Green B16F1 melanoma cells were primarily embolized in the capillary bed and had distorted morphology (∧). <b>(lower panel).</b> Green hMSC retained a regular morphology and were found both within arteries (†) and within the capillary beds (#). Images taken 10 minutes after injection of the cells. Arrows indicate direction of blood flow. Magnification 100×.</p

The potency window for each of the LPS preparations.

<p>The fraction of active cells is plotted as a function of concentration for values spanning nine orders of magnitude, as shown.</p

Activation dynamics for each of the LPS preparations and several concentrations and summary statistical comparisons.

<p>(A) As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a>, the average and ten randomly selected traces of active cells are shown, as well as Time to Peak, Peak Amp and N. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g001" target="_blank">Figure 1</a> legend for more details. Concentrations are indicated at left, and the preparation at top. The very lowest and highest concentrations are not shown here (but appear in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053222#pone-0053222-g004" target="_blank">Figure 4</a>), because virtually no cells were found to be active in the first case, and the traces are essentially identical to their nearest neighbor in the second. (B) Time-to-peak values for each LPS preparation are shown with standard deviations, across each concentration. LPS preparations are indicated with different colors as labeled in D. (C) Peak amplitude values for each LPS preparation are shown with standard deviations across each concentration. (D) The correspondence between time-to-peak and peak amplitude is shown for each LPS preparation, including all concentrations.</p

Low passage hMSC express α4 integrin and SLeX.

<p>hMSC derived from four preparations from four different donors were assayed for expression of α4 integrin and SLeX by flow cytometry. Passage 1 cells were plated overnight to recover adherent viable cells and then re-plated at 100 cells/cm². The cells were harvested when 70 to 80% confluent.</p

Clearance from the circulation of hMSC, melanoma cells and 10 µm inert beads.

<p>Inflexible inert 10 µM beads and B16F1 are cleared from circulation faster than hMSC. <b>A.</b> Values for cellular flux calculated as the average number of cells or 10 µm beads counted within vessels each minute in the CAM at 100× magnification. B. Values expressed as percentage flux were calculated as cells or beads in one minute as % of total observed in 10 minutes (n≥6).</p

The effect of blocking TNF on the NF-κB activation time courses as a function of concentration and preparation.

<p>The cosine vector distances between the average time courses in the presence and absence of sTNFRII are shown. Almost no cells were activated by UP LPS at low concentrations; hence the dashed light blue line is included for completeness but is not statistically defensible.</p