NIH/NIAID GM-CSF Modulates Global Changes in Neutrophil Gene Expression

PMNs were isolated from venous blood of healthy individuals in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases. Human PMNs (107) were cultured in RPMI/H -/+ 100 ng/ml GM-CSF at 37oC and 5% CO2 for up to 24 h as indicated. At the indicated time points, tissue culture medium was aspirated from each well and PMNs were lysed with RLT buffer (Qiagen). Purification of PMN RNA and subsequent preparation of labeled cRNA target was performed as described in Methods. Labeling of samples, GeneChip hybridization (Hu95Av2 oligonuceotide microarrays) and scanning were performed according to standard Affymetrix protocols (see http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment. These analyses allowed comparison of global gene expression in human neutrophils during spontaneous apoptosis with that in cells cultured with human GM-CSF. Keywords: time course 39 samples total: 9 time zero controls, -/+ GM-CSF at 5 time points in triplicate