Neutrophil Response to A. phagocytophilum-GSE2405_family

Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals in accordance with protocols approved by the Institutional Review Board for Human Subjects at the University of Minnesota and the National Institute of Allergy and Infectious Diseases.

PMNs (107) were combined on ice with live S. aureus (108) or with live or heat-killed A. phagocytophilum (bacteria isolated from 5x106 infected HL60 cells for a ratio of 1 infected HL60 cell: 2 PMNs, ~ 5-20 A. phagocytophilum: PMN) in wells of a 12-well tissue culture plate (pre-coated with 20% autologous normal human serum). Unstimulated control assays received either buffer (for S. aureus comparisons) or clarified HL60 lysate (for A. phagocytophilum comparisons).

Plates were centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis and incubated at 37 deg. C in a CO2 incubator 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 was performed as described in Methods.Labeling of samples, hybridization of cRNA with HU133A oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols (http://www.affymetrix.com/pdf/expression_manual.pdf ).Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment.

http://www.niaid.nih.gov/dir/labs/lhbp/deleo.htm