CEP-dependent macrophage migration in a 3D matrix. (A) Thioglycollate-induced peritoneal macrophages were isolated from WT or β₂^−/− mice and their adhesion to CEP was evaluated as described for Figure 4. (Bi) Isolated WT and β₂^−/− macrophages were labeled with green (WT) or red (β₂^−/−) fluorescent dyes. Cells were mixed in equal number and the similar amounts of cells were verified by cytospin of mixed cells. Bar represents 400 μm. (Bii) The cell number was calculated by Image Analysis Software (EVOS, Thermo Fisher) using 5 random fields. (C-E) Thrombin-treated fibrinogen forms a 3D polymerized gel in a Boyden chamber. (Ci) Labeled cells were plated on 3D polymerized fibrin in transwell inserts. Migration of macrophages was stimulated by 30 nM MCP-1 added to the top of the gel. (Cii and D) After 48 hours, migrating cells were detected by a Leica Confocal microscope The first 30 μm of the gel from the starting point (where many nonmigrated cells reside) is not shown to reduce a gradient of brightness intensity for the sample. (E) The results were analyzed by IMARIS 8.0 software and plotted. Statistical analyses were performed using Student paired t tests (n = 4 samples per group). Bar represents 500 μm.