Higher affinity and avidity of β2 integrins on ARAP3-deficient neutrophils. (A) Surface Mac-1 and LFA-1 were analyzed by FACS from flushed bone marrow cells from mock- and tamoxifen-induced Arap3^fl/flERT2Cre⁺ mice that had or had not been prestimulated with 20 ng/mL of TNFα at 37°C before being labeled with PE-conjugated anti-GR1, APC-conjugated anti–Mac-1, and FITC-conjugated anti–LFA-1. For FACS analysis, GR1-positive cells were gated and Mac-1 and LFA-1 staining was measured; results were analyzed using FlowJo v6.4.7 software. Cells from 27 tamoxifen- and mock-injected mice were analyzed in 5 separate experiments. Representative traces are shown. Gray lines represent cells from tamoxifen-induced and black lines from mock-induced Arap3^fl/flERT2Cre⁺ mice; broken lines represent unstimulated samples and full lines TNFα-stimulated samples. (B) Binding of unstimulated, bone marrow–derived neutrophils from mock-induced (WT) or tamoxifen-induced (KO) Arap3^fl/flERT2Cre⁺ mice to ICAM1-Fc in solution as a readout of affinity was measured by quantitative immunofluorescence using an FV1000 confocal microscope (Olympus) with a 60× objective, as detailed in “Analysis of β2 integrins.” Mean fluorescence intensity of 88 WT and 148 KO cells pooled from 3 separate experiments are plotted (right) and representative examples are shown (left). (C) Mac-1 and LFA-1 distribution in unstimulated neutrophils in suspension was visualized microscopically. Forty cells were analyzed for each condition in each of 3 experiments; representative examples are shown (left). Cells were scored for integrin clustering as a readout for high avidity status. Integrated values obtained from 3 separate experiments are shown graphically (see also supplemental Figure 3 for an analysis of integrin clustering using ImageJ v1.37 software). (B-C) Statistical analysis by paired t test. *P < .05; ***P < .001.