Immunoblot detection of granule proteins in neutrophils from patients with SCN. Polymorphonuclear cells (PMNs) were isolated by Dextran T-500 sedimentation (Amersham Pharmacia Biotech, Uppsala, Sweden) prior to density centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Remaining erythrocytes were lysed by hypotonic shock, and cell purity (> 95%) was determined using light microscopy. PMNs were lysed by sonification in water that contained protease inhibitors (Complete; Roche, Indianapolis, IN) and proteins were separated with SDS-PAGE (NuPAGE 4%–12%; Invitrogen, Carlsbad, CA).³  Lysate corresponding to 4 μg protein was loaded in each lane. C indicates control participants; no. 1, the patient who underwent bone marrow transplantation; and nos. 2, 3, 7, and 15, the patients with SCN. Proteins were detected with specific antibodies against the following granule proteins: HNP1–3 (BD Biosciences Pharmingen, Palo Alto, CA), lysozyme, MPO, lactoferrin (all from DAKO A/S, Glostrup, Denmark), LL-37,²  and gelatinase (Jack Cowland, Rigshospitalet, Copenhagen, Denmark). Histone H1 (Acris Antibodies, Hiddenhausen, Germany) was used as an internal control. Bound antibody was detected with horseradish peroxidase (HRP)–conjugated antibodies (goat anti–rabbit IgG [H + L] was from BioRad Laboratories [Hercules, CA] and goat anti-mouse was from Pierce Biotechnology [Rockford, IL]), followed by chemiluminescence using SuperSignal West Dura (Pierce Biotechnology).