Neutrophils deficient for PHD2 display enhanced motility in highly confined 1D and 2D microenvironments in an HIF2α-dependent manner. (A) Example of low-resolution (original magnification, ×10) imaging of WT neutrophils migrating in 1D-microchannels (3 µm width) (upper panel). Arrowheads indicate cell position. Nucleus (red) was labeled with Hoechst. Bright-field high-resolution (original magnification, ×63) image of a neutrophil migrating in a microchannel (lower panel). (B-C) Average speed of neutrophils migrating through microchannels with a width of 3, 4, or 5 µm. At least 89 cells for the WT/cKOP2 group and 127 cells for the WT/cKOP2H2 group were measured. (D) Example of low-resolution (original magnification ×10) imaging of WT neutrophils migrating in 2D-confined devices (4.5 µm width) (left panel). Higher resolution (original magnification, ×40) image of a single neutrophil migrating in a microchannel (right panel). Nucleus (red) was labeled with Hoechst. (E) Representative neutrophil tracks during random 2D migration. t indicates the period of the displayed tracking, and r represents the mean displacement ratio in this period. (F,H) Quantification of mean speed of single neutrophil trajectories migrating in 2D confined microdevices. Panel F corresponds to tracks in panel E. Data are represented as box plots (+ median), and whiskers range from the 10th percentile to the 90th percentile. (G,I) Mean square displacement of neutrophils analyzed in panels F and H, respectively. All graphs are a representative result of ≥3 independent experiments. *P < .05, Mann-Whitney U test.