Isolation and culture of human neutrophils

Neutrophils were isolated from peripheral blood of healthy subjects and COPD patients by dextran sedimentation and discontinuous plasma-Percoll gradient centrifugation, as previously described (Haslett et al., 1985; Ward et al., 1999b) in compliance with the guidelines of the South Sheffield Research Ethics Committee (for young healthy subjects; reference number: {"type":"entrez-protein","attrs":{"text":"STH13927","term_id":"1439147539","term_text":"STH13927"}}STH13927) and the National Research Ethics Service (NRES) Committee Yorkshire and the Humber (for COPD and age-matched healthy subjects; reference number: 10/H1016/25). Informed consent was obtained after the nature and possible consequences of the study were explained. Mean age in years was 61.7 ± 2.3 (n = 10) and 66.0 ± 3.6 (n = 7) for COPD and age-matched healthy subjects respectively. Ultrapure neutrophils, for Kinexus antibody array experiments, were obtained by immunomagnetic negative selection as previously described (Sabroe et al., 2002). Neutrophils were cultured (2.5 × 10⁶/ml) in RPMI 1640 (Gibco, Invitrogen Ltd) supplemented with 10% FCS 1% penicillin-streptomycin, in the presence or absence of the following reagents: GMCSF (PeproTech, Inc), N⁶-MB-cAMP (Biolog), anti-ErbB3 blocking antibody, Tyrphostin AG825 (both Sigma-Aldrich), CP-724714 (AdooQ Bioscience), Erbstatin analog (Cayman Chemicals), Pyocyanin (Usher et al., 2002) or compounds from PKIS (Published Kinase Inhibitor Set 1, GlaxoSmithKline) at concentrations as indicated.

In vitro screening of PKIS in neutrophil apoptosis assays. PKIS consists of 367 small molecule protein kinase inhibitors and is profiled with respect to target specificity (Elkins et al., 2016). In primary screen experiments, neutrophils (from five independent donors over 5 days) were incubated with each compound at 62 µM for 6 hr. Apoptosis was measured by flow cytometry (Attune, Invitrogen). Secondary screening was performed with selected compounds that accelerated neutrophil apoptosis greater than twofold in the primary screen. Compounds were incubated with neutrophils at 10 µM for 6 hr and apoptosis assessed by Attune flow cytometry.