Ficoll hypaque density

Human peripheral blood mononuclear cells (PBMCs) were obtained from normal healthy volunteers. Cells were separated from buffy coats via Ficoll-Hypaque density gradient centrifugation (Amersham Biosciences, Uppsala, Sweden). RBCs were lysed in hypotonic ACK buffer. CD4⁺ T cells were isolated from PBMCs using a CD4⁺ T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. To induce Th17-polarization, CD4⁺ T cells were stimulated with plate-bound anti-CD3 (0.5 μg/ml), anti-CD28 (1 μg/ml; BD Pharmingen), anti-IFN-c (2 μg/ml), anti-IL-4 (2 μg/ml), IL-1β (20 ng/ml), and IL-6 (20 ng/ml; R&D systems) for 72 h. Cells were cultured at RPMI (10% FBS, 1% antibiotics). Total RNA was extracted using the Tri reagent (Molecular Research Center) according to the manufacturer’s instructions. The supernatants were assayed in terms of IL-17 content. Cells were pretreated with AEBS (50–200μg/ml) for 1 day and washed. Then harvested cells were seeded at culture media with adding AEBS (50–200μg/ml) under the aforementioned Th17-polarizing conditions. The written informed consents were obtained from each participant after providing enough information and explanation about procedure. This study was approved by the Seoul St. Mary’s Hospital Institutional Review Board.

Thirty milliliters of EDTA venous peripheral blood were collected for complete blood counts (CBC), pVL, HLA typing, and T cell analyses. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation (Amersham, Sweden). Viral loads were determined using a quantitative HIV-RNA assay (COBAS AmpliPrep Taqman) with a US-FDA-approved commercial kit in an ISO-15189-certified clinical virology laboratory. CD4 counts were determined by antibody mixture commercial kit (CYTO-STAT triCHROME) in ISO-15189-certified clinical immunology laboratory. HLA-class I type was analysed by PCR-SSOP and PCR-SSP (Proimmune, United Kingdom and BGI Asia-Pacific, Hong Kong).