Cell-surface localization of elastase in AML cells. (A) Representative FACS analysis, determined using rabbit antielastase Abs and secondary anti-rabbit FITC Abs, of elastase expression on AML cell lines (U937, HL-60, and ML-1) and primary AML PB and BM cells. Staining with only secondary Abs is shown in grey. Elastase internal staining of BM AML cells was performed after cell permeabilization as described in “Materials and methods.” Perm. indicates permeabilization. (B) Immunofluorescence staining for membranal elastase and CXCR4 in primary PB AML cells. Original magnification, × 100. (C) Cell-surface expression of elastase on AML cells after incubation with SDF-1 or anti-CXCR4 Abs. BM-derived cells were treated with 200 ng/mL SDF-1 (left panel) and PB-derived cells with 10 μg/mL anti-CXCR4 Abs (right panel) for 3 hours and stained for elastase cell-surface expression. Control cells stained with only secondary anti-rabbit FITC Abs are shown with grey plain line (a); control cells stained with antielastase Abs, with bold black line (b); cells treated with SDF-1 or anti-CXCR4 for 3 hours, with thin black line (c); and cells treated with anti-CXCR4 for 16 hours, with dotted line (d).