Neutrophils from EDA-ID patients display normal NADPH-oxidase activity. Neutrophils were isolated from heparinized peripheral blood of patients and controls over isotonic Percoll, as described.⁴  (A) Cells were cultured overnight in HEPES medium (132 mM NaCl, 20 mM HEPES, 6 mM KCl, 1 mM MgSO₄, 1.2 mM K₂HPO₄, 1 mM CaCl₂, 5 mM glucose and 2% (vol/vol) human serum albumin, pH 7.4) in the presence of 10 ng/mL LPS and 50 ng/mL LBP at a concentration of 5 × 10⁶/mL, after which the IL-8 concentration was determined in the supernatant by ELISA, according to the manufacturer's instructions (Sanquin Reagents, Amsterdam, The Netherlands). Data represent the means (± SEM) of 3 independent experiments performed in duplicate. Statistical significance was determined by a Student t test. *P = .0173. (B) NADPH-oxidase activity was assessed as hydrogen peroxide production determined by an Amplex Red kit (Molecular Probes). Neutrophils (10⁶/mL) were stimulated with 1 mg/mL bare or serum-treated zymosan (STZ), 100 ng/mL phorbol myristate acetate (PMA) or PAF + fMLP (formyl-methionyl-leucylphenylalanine; both 1 μM, added simultaneously), in the presence of 0.5 μM Amplex Red and 1 U/mL horseradish peroxidase, as described.⁵  Fluorescence was measured at 30-second intervals for 20 minutes on a Spectra Fluor Plus spectrophotometer (Tecan, Zürich, Switzerland). Maximal slope of H₂O₂ release was assessed over a 2-minute interval. Data represent the means (± SEM) of 3 independent experiments performed in duplicate. No significant difference was found between patient and control cells. (C) Expression of gp91^phox and p67^phox was assessed on Western blot. Freshly isolated neutrophils of a healthy control donor (C) or an EDA-ID patient (P) were stimulated for 2 hours with 10 ng/mL TNF-α (+) or left unstimulated (−), as indicated. Afterward, cell lysates were prepared and analyzed on Western blot for the indicated targets. Cells (1.5 × 10⁶) were loaded in each lane and, in addition, ASC expression is shown as a loading control. Mouse anti-gp91^phox (clone 48) was obtained from Sanquin Reagents, rabbit anti-p67^phox was obtained from Upstate Biotechnology (Lake Placid, NY), mouse anti-ASC was obtained from MBL International (Woburn, MA). IRDye680 or 800CW conjugated secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE). The blots were analyzed with an Odyssey Infrared Imager (LI-COR).