Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells

Law, Helen KW; Cheung, Chung Yan; Sia, Sin Fun; Chan, Yuk On; Peiris, JS Malik; Lau, Yu Lung

Blood mononuclear cells were isolated from whole blood by centrifugation, using Ficoll-Hypaque gradients (Phar-macia Biotech, Uppsala, Sweden), washed, and labeled with immunomagnetic antibodies. Positive selection was performed according to manufacturer's specification (Miltenyi Biotec, Bergisch Gladbach, Germany) as in previous experiments [8] . Isolated CD14+ monocytes from the positive fraction were resuspended in RPMI 1640, supplemented with 50 IU/ml penicillin and 50 μg/ml streptomycin and 10% fetal bovine serum (Invitrogen, Grand Island, USA). Cell viability, as measured by trypan blue exclusion, was more than 95%. The purity of the isolated cells as measured by flow cytometry was consistantly between 90% to 95%.