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. 2014 Apr;95(4):677–688. doi: 10.1189/jlb.0313147

Figure 4. Analysis of heparanase expression in umbilical CB cells.

Figure 4.

(A) mRNA. MNCs were isolated on a standard Ficoll-Paque density gradient. Enrichment of MNC subpopulations was performed, as described in Materials and Methods. Monocyte purity was 95%; NK cells, 83%; macrophages, 92%; and neutrophils, 89%. Enrichment of CD4 was 89%; CD8, 82%; and CD19, 72% (n=3). Heparanase is expressed primarily in CB-derived MNCs, monocytes, and macrophages and to a lesser extent, in neutrophils and NK cells. There was little or no expression of heparanse in nonactivated T and B lymphocytes. (B) Immunostaining. Cells were subjected to immunostaining, as described in Materials and Methods. After fixation and PBS washing, cells were incubated with antiheparanase primary antibody (green). Cells were then washed extensively with PBS and incubated with AlexaFlour 488 (Cy2/Cy3-conjugated) secondary antibody, washed, and stained with PI to view the cell nucleus (red). (a) MNC; (b) neutrophils; (c) macrophages; (d) monocytes; and (e) NK cells. Original magnification, ×40. In neutrophils, CB-derived macrophages, and monocytes, heparanase is distributed uniformly in the cell membrane (B, a–d), whereas in NK cells, it appears in clusters and is also found in the nucleus (B, e).