Fig. 5.
Unidirectional aggregation of 3T3x cells. (a) Circular pattern of adhesive strips (gray areas) on a nonadhesive substrate (bright areas) generated by evaporating a thin layer of NiCr onto a Sylgard 184 surface with a bar pattern electron microscope grid as a mask (r = 1.25 mm). (Bar, 1 mm.) (b) According to the basic rationale, the outermost cell aggregates on each strip must be located a distance Ra away from each end. The two circular arcs indicate these locations. (c) Example of aggregates of hyperscattering cells located along the predicted aggregation arcs as seen in brightfield microscopy. (d) Quantitative evaluation of the circular aggregation arcs by matching them to two circles of radius r. The shift between the circles is 2Ra. Obviously, Ra is much larger than one cell diameter, suggesting that there is a long-range attraction between the aggregating cells. (e–g) Increase of visibility and accuracy of the aggregation arcs by image averaging. (e) Unprocessed image. (f) Superimposition of e with its own mirror image (self-mirrored image). (g) Image average of all five test fields of a test substrate, each processed as in f.(h) Visualization of aggregation arcs in the case of low-level scattering of ingested particles. Arc formation of cells that had ingested 0.1-μm fluorescent latex particles. Even though the particles are too small to be detected in brightfield micrographs, the self-mirrored fluorescence micrograph shows the location of the outermost aggregates away from the ends of the adhesive strips. (i and j) Visualization of aggregation arcs in the case of particle-free 3T3x cells. (i) Hoechst staining of a field of particle-free cells. (j) LysoTracker staining of the same field reveals an aggregation arc formed by lysosome-rich cells. (k) Coomassie-blue-stained 3T3x cells that had formed aggregation arcs on the large and wide-spaced strips derived from a 100-mesh grid during the 5 days after they had ingested white diamond particles. White arrows point to the aggregates of particle-rich cells that constituted parts of the arc. Obviously, the sheet of particle-rich, aggregated cells did not retract under some kind of tension away from the ends of the strip but were embedded in a much larger cell sheet that covered the entire strip. (Bar, 150 μm.)