Human monocyte-derived macrophages obtained from peripheral blood mononuclear cells
Treatment protocol
Just before the experiment with cytokines the medium was changed to DMEM containing 0.5% of pooled human serum and the cells were stimulated with IL-1 (15 ng/ml), or IL-6 (25 ng/ml) for the period of 4 h. These rather low concentrations of cytokines that may occur in vivo were selected in preliminary trials as sufficient to stimulate the expression of manganese superoxide dismutase (MnSOD).
Growth protocol
Human monocyte-derived macrophages (hMDMs) were obtained from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human blood of 7 healthy donors using a Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient. Cells from each donor were split into three aliquots to stimulate them later with a mock stimulant, IL-1 and IL-6 and plated at 4x107 in PrimariaTM 25cm2 cell culture flasks with vented caps (Becton Dickinson, Franklin Lakes, NJ, USA) in RPMI1640 (Invitrogen) supplemented with 2mM L-glutamine, 50 g/ml gentamycin (Sigma), and 10% pooled heat-inactivated human AB serum. After 24 h, non-adherent PBMCs were removed by washing with complete medium, and adherent cells were cultured in this medium for 7 days with fresh medium changed every 2 days. The MDMs phenotype was controlled, after non-enzymatic detachment of cells, by immunofluorescent staining of CD14 (clone: TÜK4, DakoCytomation Denmark A/S, Glostrup, Denmark), CD16 (clone: DJ130c, DakoCytomation), CD11b (clone: ICRF44, Becton Dickinson and Co., Franklin Lakes, USA), and CD209 (clone: DCN46, Becton Dickinson) and subsequent flow cytometry analysis. The routine procedure used in our laboratory yields cells positive in at least 90% for the first three markers and less than 1% for CD209. It should be added that the adherent cells acquire typical macrophage morphology and extensive phagocytic activity against live Staphylococcus aureus and apoptotic neutrophils. Resting (non-stimulated) cells do not produce proinflammatory cytokines: IL-1, TNF-a or IL-6.
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA was isolated from unstimulated (control) and cytokine-stimulated cells by Chomczynski and Sacchi procedure.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following labeling, samples were hybridized to the GeneChip Test3 array (Affymetrix, Santa Clara, CA) for quality control. Fragmented cRNA (15 μg) was used for hybridization to the GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). Arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidic Station 400 (Affymetrix, Santa Clara, CA) according to standard protocol of the manufacturer.
Scan protocol
The arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
