Motility assays of cytokine-treated cells. (A) Random migration assay across a multiporous filter. Nonadherent cells were recovered on day 7 of GM-CSF (middle) or IL-3 (right) treatment and placed in the upper wells of a modified Boyden chamber in triplicate. After 90 minutes of incubation at 37°C, the filter was Giemsa stained and the migrated cells to the underside were counted in four randomly chosen HPFs (original magnification × 400). Bars represent the numbers of migrated cells per HPF (mean ± SD). Control, unstimulated cells. (B) Morphologic polarization activity of myeloid cells. Day 7 GM-treated and IL-3–treated nonadherent cells were either preincubated with GM-CSF for 30 minutes before assay (priming [+]) or left unprimed until assay (priming [−]). Cells were challenged by PBS, fMLP, or C5a for 10 minutes to evoke polarization. Bars show the polarization activities of cells calculated as in Materials and Methods, after being challenged by PBS (□), fMLP (), or C5a (▪). (C and D) Polarized cells on C5a stimulation. More than 50% of the GM-treated cells (priming [+]) underwent characteristic morphological changes (C; original magnification × 150), while the IL-3–treated cells contained much fewer polarized cells with milder alterations in shape, even with priming (D; original magnification × 150).