CD14+ peripheral blood-derived monocytes were isolated from leukopacks from healthy donors by counterflow centrifugal elutriation. Fresh monocytes were cultured in 6-well tissue culture plates at 2-3 ยด 106/ml in complete RPMI 1640 supplemented with 20 mM glutamine, 2% heat-inactivated human AB serum, 100 ug/ml penicillin, and 100 g/ml streptomycin. For generation of DC, recombinant human (rh) IL-4 and rhGMCSF were added to the culture at 50 ng/ml on days 1, 3, and 5 of culture. Cells were exposed to the pathogens on day 8 of culture, and 16 hours later were harvested and used for RNA preparation. Because of donor-to-donor differences, human experimental systems can be prone to considerable variability. Therefore cells from a total of 7 donors were prepared and infected independently. The RNA from all 7 donors was extracted separately; equivalent amount of RNA from each donor was pooled, and used for subsequent labeling and hybridization. We assessed the variability associated with the preparation of targets and processing of microarrays by replicating this procedure with another set of RNA from the same donors. Keywords = Infectious diseases, Parasites, Infection, Innate immunity
