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. 2016 Aug;186(8):2105–2116. doi: 10.1016/j.ajpath.2016.04.001

Figure 6.

Figure 6

Adhesion and spreading of WT and Mac-1–deficient macrophages. Peritoneal macrophages isolated from a 3-day inflamed peritoneum of WT and Mac-1−/− mice were plated on Permanox dishes (A–C) or glass coverslips (D and E). A: Macrophages were allowed to adhere for 10, 20, and 30 minutes at 37°C. Nonadherent cells were removed, and adherent cells were labeled with Alexa Fluor 546 phalloidin and DAPI. Ten randomly selected fields were photographed for each condition, and adherent cells were counted with ImageJ software (NIH, Bethesda, MD; http://imagej.net). B: Representative images of spreading of WT and Mac-1–deficient macrophages. C: Percentage of spread cells was determined from the five images of adherent cells. D: Fusion of inflammatory peritoneal macrophages isolated from WT and Mac-1−/− mice plated on glass coverslips for 2 hours was induced with 10 ng/mL IL-4. After various periods of time, cells were fixed with 2% paraformaldehyde and stained with Rhodamine phalloidin. Representative images of cells 2, 6, 12, 24, and 48 hours after plating. E: The total number of actin-based protrusions was counted and divided by the total number of cells in the field. Data are expressed as means ± SD. n = 10 fields with approximately 300 cells per image (A); n > 200 cells in 3 to 5 randomly selected fields of 3 separate experiments (E). ***P < 0.001. Scale bar = 20 μm. Original magnification: ×20 (B and E). WT, wild-type.