Ficoll hypaque plus

Monocytes were isolated from HV peripheral blood by centrifugation on Ficoll-Hypaque Plus (Amersham Biosciences) and adherence, as we have described before (13 (link)).The composition of this adherent population of cells was analyzed by FACS. Once seeded, adherent cells were treated with 100 μM of DMOG two hours before 24 h of 10 ng/mL LPS stimulation. Cytokine productions on supernatant and cell surface markers were checked by CBA and cytometric analysis.

Fresh (not frozen) PBMCs were isolated with Ficoll-Hypaque plus (Amersham Bioscience, Uppsala, Sweden) and stained with anti-CD8 antibody-coated microbeads (Miltenyi Biotech, Auburn, CA) in accordance with the manufacturer’s recommendations. Positive selection of CD8 lymphocytes was performed with the Miltenyi Biotech MACS system. Short-term (3-week) CD8 T-cell cultures were set up as previously described (48 (link)). Briefly, sorted CD8 lymphocytes were plated at a 5:1 ratio with 1 μM peptide-pulsed (for >1 h), irradiated T2 cells (CRL-1992; ATCC, Manassas, VA) that were washed free of excess peptide before being mixed with CD8 T cells. CD8 T-cell lines were fed with AIM V medium (Gibco) supplemented with 14% human AB serum (Nabi, Miami, FL, or Gemini, Woodland, CA), 16% MLA-144 supernatant (75 (link)), 10 U/ml recombinant IL-2 (BD), 1% l-glutamine (Gibco), 0.5% β-mercaptoethanol (Sigma, St. Louis, MO), 1% HEPES (HyClone, Logan, UT) every 3 to 4 days and restimulated with fresh peptide-pulsed T2 cells weekly for only 3 weeks. This culture method has been optimized so as not to skew the TCR repertoire from that present in vivo (43 (link), 58 (link), 60 (link)).

After overnight fasting, 30 ml venous blood was drawn into an EDTA-coated vacutainer tube. The blood was diluted two-fold with phosphate buffered saline (PBS, pH 7.4). 4 ml diluted blood was layered onto 4 ml Ficoll-Hypaque Plus solution (Amersham Pharmacia Biotech AB, Sweden) and centrifuged at 2000 rpm for 20 min at room temperature. PBMC were removed from the plasma-Ficoll interface and washed three times in PBS to remove platelets, Ficoll-Hypaque and plasma. Collected PBMC were cryopreserved at -80 °C and used later for quantification of mRNA expression.

Monocytes were purified from buffy coats of healthy donors (Karolinska University Hospital) by using a RosetteSep monocyte purification kit (Stem Cell Technologies) and Ficoll-Hypaque Plus (Amersham Biosciences) gradient centrifugation. Human DCs were seeded at 0.8 × 10⁶ to 1.5 × 10⁶ cells/ml in R10 (RPMI 1640, 2 mM l-glutamine, 10% fetal bovine serum [FBS]) supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF; 40 ng/ml) and IL-4 (40 ng/ml) (both from PeproTech) for 6 days. Cells were given fresh media and cytokines at a ratio of 1:1 on day 4 and cultured until day 6. The human DC phenotype was assessed by examination of CD11c and CD1a expression via staining with allophycocyanin (APC)-conjugated mouse anti-human CD11c and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD1a (BD Pharmingen). DCs used in these experiments were above 90% CD1a⁺/CD11c⁺.

Peripheral blood was collected from the study participants at baseline and during the 1^st and 4^th weeks. Peripheral blood mononuclear cells (PBMCs) were isolated from white blood cells by the standard Ficoll-Hypaque Plus (Amersham Biosciences, Uppsala, Sweden) density gradient separation technique. RNA was purified from the PBMCs using the RiboPure™ Kit (Ambion, Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. The RNA integrity was assessed by agarose electrophoresis. RNA samples with an A260:280 ratio > 1.8 were selected for the microarray. The isolated RNA was used as the template for one round of reverse transcription to generate cDNA with the ThermoScript RT-PCR System.

Blood was dispensed into polypropylene tubes (BD Falcon™) and diluted 1:2 in sterile filtered phosphate-buffered saline (PBS). This solution was added to polypropylene tubes (BD Falcon™) containing Ficoll-Hypaque PLUS™ (Amersham Biosciences) (1:3). Cells were separated by centrifugation for 30 min at 900 x g 20°C, and the PBMC ring formed between the Ficoll interface and plasma was collected. Cells were washed twice by centrifugation and resuspended in DMEM medium (SIGMA™) supplemented with 10% FBS and 1% gentamicin-amphotericin (GIBCO^®). Cells were counted in a Neubauer chamber using Trypan Blue dye (Sigma™).