Figure 2.
Figure 2. Characterization of cultivated BM EPCs and dysfunctional BM EPCs in corticosteroid-resistant ITP patients compared with corticosteroid-sensitive ITP patients and healthy controls. (A) The typical EPC phenotype of cultivated BM EPCs was confirmed by demonstrating the positive expression of CD34, CD309, and CD133 at day 7 in culture by flow cytometry (full line). Aliquots of isotype-identical antibodies served as negative controls (dotted line). (B) Representative images (left panel) for cultivated BM EPCs at day 7 in culture among patients with corticosteroid-resistant ITP, corticosteroid-sensitive ITP, and healthy controls (Normal). Typical BM EPCs were characterized by double-positive–stained (merged in yellow) with DiI–Ac-LDL (red) and FITC–UEA-I (green) (original magnification ×10). Quantification (right panel) of double-positive–stained cells (merged in yellow) with DiI–Ac-LDL (red) and FITC–UEA-I (green) at day 7 in culture among the corticosteroid-resistant ITP, corticosteroid-sensitive ITP, and healthy control groups (original magnification ×10). The data were expressed as the means ± SEM. (C) Tube formation was inspected on an inverted light microscope. The relative tube length per field of view was analyzed by Image Proplus by counting 3 random fields per sample. Representative images (left panel) and quantification (right panel) of tube formation (pixels of tubes per field of view) by cultivated BM EPCs at day 7 in culture (original magnification ×4). (D) The migrated cells (blue) were fixed and stained with crystal violet. Cell images were obtained on a phase-contrast microscope (Olympus) and counted in 3 random fields per sample. Representative images (left panel) and quantification (right panel) of the transwell migration assays of cultivated BM EPCs at day 7 in culture (original magnification ×10). The numbers of migrated BM EPCs per field of view were compared among patients with corticosteroid-resistant ITP, corticosteroid-sensitive ITP, and healthy controls. Three power fields were randomly counted and averaged per sample. Subject variables were compared using the Mann-Whitney U test for continuous variables. All P values <.05 were considered significant and were provided in the figure.

Characterization of cultivated BM EPCs and dysfunctional BM EPCs in corticosteroid-resistant ITP patients compared with corticosteroid-sensitive ITP patients and healthy controls. (A) The typical EPC phenotype of cultivated BM EPCs was confirmed by demonstrating the positive expression of CD34, CD309, and CD133 at day 7 in culture by flow cytometry (full line). Aliquots of isotype-identical antibodies served as negative controls (dotted line). (B) Representative images (left panel) for cultivated BM EPCs at day 7 in culture among patients with corticosteroid-resistant ITP, corticosteroid-sensitive ITP, and healthy controls (Normal). Typical BM EPCs were characterized by double-positive–stained (merged in yellow) with DiI–Ac-LDL (red) and FITC–UEA-I (green) (original magnification ×10). Quantification (right panel) of double-positive–stained cells (merged in yellow) with DiI–Ac-LDL (red) and FITC–UEA-I (green) at day 7 in culture among the corticosteroid-resistant ITP, corticosteroid-sensitive ITP, and healthy control groups (original magnification ×10). The data were expressed as the means ± SEM. (C) Tube formation was inspected on an inverted light microscope. The relative tube length per field of view was analyzed by Image Proplus by counting 3 random fields per sample. Representative images (left panel) and quantification (right panel) of tube formation (pixels of tubes per field of view) by cultivated BM EPCs at day 7 in culture (original magnification ×4). (D) The migrated cells (blue) were fixed and stained with crystal violet. Cell images were obtained on a phase-contrast microscope (Olympus) and counted in 3 random fields per sample. Representative images (left panel) and quantification (right panel) of the transwell migration assays of cultivated BM EPCs at day 7 in culture (original magnification ×10). The numbers of migrated BM EPCs per field of view were compared among patients with corticosteroid-resistant ITP, corticosteroid-sensitive ITP, and healthy controls. Three power fields were randomly counted and averaged per sample. Subject variables were compared using the Mann-Whitney U test for continuous variables. All P values <.05 were considered significant and were provided in the figure.

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