NIH/NIAID Chronic Granulomatous Disease Neutrophils

Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals (Healthy1, Healthy2, Healthy3, and Healthy4) or patients with X-linked chronic granulomatous disease (XCGD1, XCGD2, XCGD3, XCGD4, XCGD5, and XCGD6). The studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases, and the Institutional Review Board for Human Subjects at the University of Iowa. All studies were conducted according to Declaration of Helsinki principles. PMNs (107) were combined with or without IgG and C3bi-coated latex beads (8 x 107) in wells of a 12-well tissue culture plate (pre-coated with 20% normal human serum) and centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis. For the purpose of these studies, activated or "Stimulated" PMNs are defined as those that have been stimulated by phagocytosis of IgG- and C3bi-coated latex beads. "Control" PMNs are defined as resting cells or those left unstimulated throughout the time-course. Following centrifugation, plates were incubated at 37 deg. C in a CO2 for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target (12 g) was performed as described in Methods. Labeling of samples, hybridization of cRNA with Hu95Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols. Gene expression in healthy control and XCGD individuals was always compared at the same time points after phagocytosis Keywords = HUMAN PMN CHRONIC GRANULOMATOUS DISEASE NEUTROPHILS Keywords: parallel sample