Human Monocytes Derived Macrophages Isolation and siRNA transfection

Blood was obtained from human volunteers by venipuncture of an antecubital vein using sterile technique following informed consent. These volunteers were separate from the volunteers for the fiberoptic bronchoscopy studies. The University of Pittsburgh Institutional Review Board approved the study. Briefly, blood was anticoagulated using citrate phosphate dextrose solution. Approximately 30 ml were laid on Ficoll-Paque Plus for gradient centrifugation and the mononuclear cell layer at the interface was harvested. Mononuclear cells were washed three times with PBS, then resuspended in RPMI 1640, and plated onto petri-dishes. The cells were incubated at 37°C, 5% CO2 for 1 hour. Plated cells were then washed with RPMI 1640 containing fresh media with 10% autologous serum plus recombinant human-M-CSF at 50 ng/ml. Cells were incubated for 5 days with one round of media change. Following differentiation, Human Monocyte Derived Macrophages (HMDM) were detached from the petri-dishes by placing the dishes on ice for 1 hour. The cells were counted, aliquoted and centrifuged down at 400g for 5 minutes. The cells were then resuspended in 100 μl of Amaxa Human Macrophage Nucleofector^® Solution containing 800 nM of control siRNA or IRF-7 siRNA and transferred to a cuvette. The cells were transfected by electroporation in Nucleofector I device with program Y-10 (Lonza, Walkersville, MD). Cells were retrieved by rinsing the cuvette with 500 μl of Aim-V medium with 10% autologous serum and transferred to 12-well plates for incubation. Twenty-four hours later, the media was changed and HMDM were exposed to ultrapure LPS (E. coli 0111:B4, 100 ng/ml, List Biological Laboratories, Cat# 421, Lot# 4217A1) for 24 hours. The cell culture supernatants were harvested for cytokine analysis by ELISA (Duoset, R&D systems) and the cells were lysed in Trizol for RNA extraction or lysed with complete protein lysis buffer for immunoblotting. RNA was extracted using Qiagen miRNeasy mini kit and cDNA was synthesized using Invitrogen SuperScript III Reverse Transcriptase kit. Real time quantitative PCR was performed according to Applied Biosystems 7900HT Fast Real-Time PCR System manufacturer instructions.