Hck is required for 3- but not 2-dimensional macrophage migration. (A) The gates used for flow cytometry sorting of wt or Hck^−/− CD11b⁺ and F4/80⁺ BMDMs. Hck^−/− BMDMs (gray open histograms) express the same levels as wt (black open histograms) of the differentiation markers CD11b and F4/80 antigens. Closed histograms represent cells alone. (B) Log₁₀ expression levels of genes in resting wt and Hck^−/− BMDMs in M1 (□) or M2 (■) polarization. Results are expressed as the ratio of the expression level in Hck^−/− BMDMs versus wt BMDMs. (C-D) Defective migration of Hck^−/− BMDMs through Matrigel matrix. (C) BMDMs were cultured on commercial Matrigel transwells for 24 hours and cells that reached the lower face of the membrane were counted. Mean value of migrating wt cells (860 ± 723 cells; n = 3) was set arbitrarily to 100%. (D) Quantification of 3D cell migration experiments performed in triplicate. Mean value of migrating wt cells (38% ± 14.3%) was set arbitrarily to 100% (n = 4). (E-F) BMDMs from wt and Hck^−/− mice have similar 2D-migration capacity tested with 2 in vitro assays. (E) Scraped area replenishment assay. Replenishment of the scraped area was measured at the indicated time points using the ImageJ software. (F) wt and Hck^−/− BMDM 2D-migration through uncoated or fibronectin-coated transwells. BMDMs that reached the lower face of transwells were counted. Mean value of migrating cells (n = 3) was set arbitrarily to 100%; *P < .05; ***P < .001.