Fig 1: Migration in vitro of ST8SiaIV−/− neutrophils toward CXCL1 and fMLP is compromised compared with WT neutrophils(A and B) Chemotaxis of neutrophils from WT and ST8SiaIV−/− mice was evaluated in a microfluidic-based microchannel assay, as described in STAR Methods. Cell migration toward fMLP (A) and CXCL1/KC (B) gradients was visualized and recorded by time-lapse live microscopy. The speed, velocity, and persistence of cells was computed from individual cell tracks (n = 191 and n = 234 from WT and ST8SiaIV−/− mice, respectively). Data are expressed as mean ± SD using cells from three mice per group from each of four experiments. ***p ≤ 0.001; ****p ≤ 0.0001; ns, not significant (Welch’s t test).(C) Bone marrow neutrophils from WT and ST8SiaIV−/− mice were isolated and stained for CD11b, Ly6G and CXCR1 and were analyzed by flow cytometry. Dot plots represent data from five mice of each strain.(D) Bone marrow neutrophils were isolated and incubated with CXCL1/KC (100 ng/mL), washed five times with PBS, lysed in RIPA buffer, and analyzed for boundCXCL1/KC by ELISA. Data are expressed as mean ± SEM (n = 3 mice per group) from each of three experiments. ****p ≤ 0.0001 (two-tailed t test).(E) Bone marrow neutrophils were isolated and incubated with 1 µM fMLP for the indicated times and collected and lysed in RIPA buffer, and equal amounts of protein (25 µg) from each lysate was separated on SDS-PAGE and analyzed for the amount of phosphorylated and total ERK as indicated in STAR Methods. Histogram shows the ratio of phosphorylated to total ERK at each time point. The immunoblot is representative of results obtained from three independent experiments.