Fluid flow primes leukocytes to resist detachment by inducing and maintaining F-actin in upstream anchors. (a) Detachment assays were performed with vehicle-, CytoD-, or LatB-pretreated U937 cells. Cells were adhered to a VCAM-1–coated surface. Fluid flow was introduced, either directly with 4 dynes/cm² (open symbols) or in a stepwise fashion (1, 2, and 4 dynes/cm²; solid symbols), and cells remaining adherent were quantified (means ± SD, n = 8; two IEs; *, P < 0.05; **, P < 0.005; ***, P < 0.0001 direct versus stepwise shear). (b) Increase in fluid flow induces F-actin accumulation in anchors. Representative confocal images of a Lifeact-GFP–transfected U937 cell exposed to fluid flow (shear stress: 2 and then 4 dynes/cm²). GFP accumulation is shown within anchors (solid arrowheads) and at their insertions (open arrowheads). The numbers at the bottom of the images refer to the height of each image above the adhesion surface. (c) Removal of fluid flow reduces F-actin accumulation in anchors. U937 cells adhered to VCAM-1 were exposed to fluid flow (2 dynes/cm² for 2 min), and then flow was stopped. Sequential z stacks of the same cell were obtained before (0 s) and after (300 s) flow was stopped. (left) Top and side view projections of a representative cell demonstrate that upstream anchors (solid arrowheads) diminished after cessation of flow. (right) Sequential images just above the adhesion surface illustrate progressively decreasing F-actin at anchor insertions (open arrowheads). See Videos 7 and 8. Arrows indicate the direction of fluid flow. Bars, 10 µm.