Figure 2.

Requirement of Mac-1 in mediating macrophage fusion. A: Time course of IL-4–induced fusion of WT and Mac-1–deficient macrophages. Cells were isolated from the peritoneum 3 days after TG injection and plated on Permanox dishes with the use of a procedure described in the Materials and Methods. After 24, 48, and 72 hours, cells were fixed and treated with Wright stain. B: The time-dependent fusion rates were determined for WT and Mac-1–deficient macrophages from Wright stain. C: The number of nuclei in MGCs formed after 72 hours from WT and Mac-1–deficient macrophages. D: Representative images of WT and Mac-1–deficient macrophages labeled with PKH26 (red) or PKH67 (green) and then cultured with 10 ng/mL or without IL-4 for 48 hours. Microscopic images were acquired in the green and red channels and overlaid. E: Colocalization (yellow) of two dyes that indicate the MGC formation was determined using ImageJ program (NIH, Bethesda, MD; http://imagej.net). Data are expressed as means ± SD (B and E) or percentage of a total number of counted MGCs (C). n = 5 to 10 experiments with approximately 400 cells counted in 5 to 10 representative low-power microscopic fields in each experiment (B); n = 25 to 130 MGCs (C); n > 10 experiments each with 5 random fields per slide (E). **P < 0.01, ***P < 0.001. Scale bars: 100 μm (A); 200 μm (D). Original magnification: ×20 or ×40 (B). MGC, multinucleated giant cell; TG, thioglycollate, WT, wild-type.