Length of incubation period affects the regulation of macrophage chemotaxis by TGF-β1. Assays were performed in transwell cell culture chambers with polycarbonate filters (24-well, 8-μm pore, Corning Costar). Raw 264 cells preincubated with or without 5 ng/mL TGF-β1 for 1 hour or 24 hours were washed, resuspended in 200 μL DMEM-12, and then added to the upper reservoir. One milliliter of serum-depleted DMEM-F12 was added to the lower reservoir with or without chemotaxis reagents (1 μg/mL LPS, 50 U/mL IFN-γ, 100 nM fMLP, and 5 ng/mL TGF-β1). After 6 hours, cells remaining on the upper membrane were scraped off, and cells on the lower membrane surface were stained with (A) Giemsa and (B) counted under a microscope. The results are means ± SE of 3 independent experiments. Each value was compared to the corresponding control at time 0 (**P < .01).