ARAP3 controls integrin-dependent chemotaxis. Bone marrow–derived neutrophils were prepared from mock-induced (WT) and tamoxifen-induced (KO) Arap3^fl/flERT2Cre⁺ mice as detailed in “Neutrophil purification.” (A) Cells were allowed to adhere to glass-bottomed dishes before dishes were filled with buffer and a micropipette filled with 1μM fMLF in buffer was inserted. A slow, constant flow of fMLF was initiated using a microinjection system, and cell movements were followed by phase-contrast time-lapse imaging over 30 minutes, taking an exposure every 30 seconds using a Zeiss Axiovert 200 microscope with a 32× objective and Axiovision v3.0 software. The needle was moved away from the cells when they threatened to migrate into the micropipette and potentially block it (WT cells only). Individual cells were tracked using the manual tracking plug-in for ImageJ. The final images of representative videos containing the tracks are shown. For the WT cells, asterisks indicate previous positions of the micropipette tip. (B-D) Chemotaxis in Dunn chambers. Cells were allowed to migrate toward 300nM fMLF in Dunn chambers and their movements were recorded by time-lapse imaging using 5× magnification. (B) Pooled tracks of individual cells obtained from experiments carried out on 3 separate days with separate cell preparations were plotted using the Ibidi chemotaxis tool plug-in for ImageJ. The source of fMLF is at the bottom. The tracks were analyzed using the chemotaxis tool's statistics feature. Accumulated and Euclidean distances (C) as well as cell speed (D) of WT and KO cells are plotted (mean ± SEM). Data were analyzed using t tests (Mann-Whitney), and differences were found to be statistically significant (P < .001). (E-G) Neutrophils from mock-induced (WT) and tamoxifen-induced (KO) Arap3^fl/flERT2Cre⁺ mice were used in chemotaxis assays in the EZ-TAXIS chamber. Cells were lined up at the bridge of an EZ-TAXIS chamber and allowed to migrate toward 3μM fMLF while being followed by time-lapse imaging using a BD pathway reflection microscopy system. (E) Cell movements were tracked and analyzed as for Dunn chamber chemotaxis; cell tracks from pooled cells tracked in 2 separate experiments are shown. (F-G) Tracks were analyzed using the statistical features of the Ibidi chemotaxis tool, and plotted results show pooled data from 3 experiments performed on separate days with separate cell preparations. Accumulated and Euclidean distances traveled by WT and KO cells (left) and their cell speeds were significantly different, as shown by t test (P < .001). (H) Transwell chemotaxis. Flushed bone marrow cells from tamoxifen- and mock-induced Arap3^fl/flERT2Cre⁺ mice were placed in the top well of polycarbonate transwells with 3-μm pores. The bottom chambers contained the indicated concentrations of fMLF. Cells that migrated through the filters in 40 minutes were counted. Graph shows pooled data (mean ± SEM) obtained from 3 individual experiments performed on separate days with separate cell preparations. Stimulation with fMLF caused significant activation of chemotaxis (P < .0001), whereas there was no significant difference between WT and KO cells (P = .5098; 2-way ANOVA with Bonferroni post test). (J-K) Chemotaxis in a 3D collagen matrix. Bone marrow neutrophils from tamoxifen-induced (KO) and mock-induced (WT) Arap3^fl/flERT2Cre⁺ mice were mixed with type I collagen in custom-made chambers. After allowing the collagen to polymerize, creating a 3 mg/mL matrix, gels were overlaid with 0.5μM fMLF and the chambers sealed. Gradients were allowed to develop and chemotaxing cells were followed by time-lapse imaging for 15 minutes, taking an exposure every 30 seconds. Cells were tracked through the frames using the manual tracking and chemotaxis tools plug-ins in ImageJ v1.37. The tracks in panel J represent pooled tracks from randomly chosen cells from 3 independent runs performed in one day. The graph in panel K represents the pooled accumulated and Euclidean distances traveled by the randomly tracked cells from 3 independent runs of WT and KO cells in experiments performed on 3 separate days with separate cell preparations (mean ± SEM). Data were analyzed using t test (Mann-Whitney); **P < .01.