Flow cytometric analysis of immunoflourescent staining of blood mononuclear cells from untreated C57BL/Ka mice or from mice receiving G-CSF for 5 days. (A) through (D) show two-color analyses of mononuclear cells from untreated mice stained with conjugated MoAbs: PE — anti–Mac-1 versus APC–anti-TCRαβ (A), PE — anti–Mac-1 versus FITC–anti-B220 (B), FITC–anti-CD4 and FITC–anti-CD8 versus APC–anti-TCRαβ (C), and FITC–anti-CD4 and FITC–anti-CD8 versus PE–anti-NK1.1 after gating on TCR αβ⁺ cells (D). Boxes enclose either Mac-1⁺, TCR αβ⁺, or B220⁺ cells, and the percentage of cells in each box is shown. The vertical line in (C) shows the threshold for gating TCRαβ⁺ cells. Conjugated isotype-matched irrelevant antibodies stained less than 2% of cells. (E) through (H) show the same staining analyses for mononuclear cells obtained from G-CSF–treated mice. (I) shows the two-color analysis of low-density cells from G-CSF–treated mice after staining for CD4 and CD8 versus TCRαβ receptors. (J) shows the analysis of CD4 and CD8 versus NK1.1 receptors after gating on TCRαβ⁺ cells from (I). (K) and (L) show one-color analysis of unfractionated mononuclear cells from untreated (solid line) or treated (dashed line) mice stained with FITC–anti-CD4 or FITC–anti-CD8 antibodies, respectively.