Surface expression of CXCR4. (A) L-selectin cross-linking increased lymphocyte surface expression of CXCR4. PBMCs were preincubated with isotype control antibody (IgG1), LAM1-14, or LAM1-3 at 10 μg/mL on ice for 30 minutes, washed, then followed by cross-linking with goat antimouse antibody (20 μg/mL) at 37°C for 1 hour. CXCR4 expression was measured by staining cells with PE-conjugated anti-CXCR4 (12G5) after blocking nonspecific binding sites with mouse IgG (50 μg/mL). Means ± SEM of relative expression (% of control) from 3 separate experiments are shown. *P < .05 compared to IgG1. (B) Representative histograms demonstrating CXCR4 expression after cross-linking using isotype control IgG1 (dotted line) or LAM1-3 (solid line). (C) Lymphocyte surface CXCR4 was increased after in vitro incubation. Freshly isolated human PBMCs were incubated in vitro for 1 hour at 4°C or 37°C. Intact or saponin-permeabilized cells were assessed for CXCR4 expression by immunofluorescence staining and flow cytometric analysis. Lymphocytes were differentiated from monocytes by light scatter characteristics.