Ficoll paque histopaque 1077

Blood from Buffy Coat units (50–70 mL) were obtained from healthy volunteers. Blood was centrifuged in Ficoll-Paque (Histopaque-1077; Sigma-Aldrich) for 35 min at 1.500 rpm and room temperature. The interphase containing peripheral blood mononuclear cells (PBMCs) was collected and washed in PBS. The desired TCR γδ⁺ T lymphocytes were labeled by incubation with hapten-conjugated anti-TCRγδ monoclonal antibody (Miltenyi Biotec GmbH), according to the manufacturer’s instructions. Further, cells were labeled with FITC-conjugated anti-hapten monoclonal antibody coupled to magnetic microbeads. The cell suspension was loaded onto a LS magnetic column (Miltenyi Biotec) and TCRγδ⁺ T lymphocytes were positively selected. γδ⁺ T lymphocytes were subsequently collected from the columns, following the manufacturer’s instructions, and resuspended in serum-free culture medium (OpTmizer-CTS) supplemented with 5% fetal bovine serum and 2 mM L-glutamine (Thermo Fisher Scientific) and 70 ng/mL of interleukin-2.

Mononuclear cells were separated by density gradient centrifugation [40 (link)]; whole blood was centrifuged at 300 g for 15 min, Buffy coats were diluted 1:1 in warmed Dulbecco’s phosphate-buffered saline (D-PBS; Sigma, UK) and over-layered onto Ficoll-Paque (Histopaque-1077; Sigma, UK), then centrifuged at 400 g for 30 min. Contaminating red cells were removed using erythrocyte lysis buffer (ELB) containing 0.15 M ammonium chloride [41 (link)]. Cells were then labelled with anti-human CD14 conjugated to magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions and passed over magnetic columns to isolate CD14⁺ cells [31 (link), 42 (link)]. Following this process, numbers of dead cells were negligible (determined by Trypan blue). Cells were suspended in RPMI with glutamine (Sigma UK) supplemented with 10% FCS (Sigma), containing 200 U/ml penicillin (Sigma) and 200 mg/ml streptomycin (Sigma) at 5–20 x 10⁵cells/ml (1–4 x 10⁵cells/well) in 96-well plates (Costar) in triplicate for biochemical assays or in 24-well plates (Costar) for RNA extraction (3–4 × 10⁶ cells/well). Cells were incubated at 37 °C in 5% CO₂, medium being replaced every 2 days, until cells achieved 80% confluence and they had matured into monocyte-derived macrophages (MDM), at 6–12 days culture.

The effect of the dietary intake of High OC/OL-EVOO on apoptosis was also investigated by TUNEL Assay in isolated peripheral blood mononuclear cells (PMBC) isolated from peripheral blood from the CLL patients during the dietary intervention. Approximately 20 ml of venous blood from the patients with CLL were obtained in a heparinized syringe (heparin, 90 IU/10 ml blood). The blood was overlaid on Ficoll–Paque (Histopaque 1077, Sigma, Aldrich Company Ltd, Dorset, UK) at a 2:1 ratio and centrifuged at 400g for 35 min at 20°C. PBMCs were collected from the interface and washed with PBS buffer (GIBCO Invitrogen Corporation, Carlsbad, CA, USA) three times to remove the plasma and the Ficoll, according to the manufacturer’s instructions. Isolated PBMC were transferred to the slides and fixed in 4% formaldehyde in PBS (pH 7.4) for 30 min at 4°C. Then the cells were washed twice in PBS and permeabilized in PBS containing 0.2% Triton x-100 for 30 min at room temperature. After washing with PBS, TUNEL was performed according to the manufacturer’s instructions (Biotium, Inc. Fremont, CA USA). DAPI staining at concentration 0.5 μM (Abcam UK) was performed incubating the slides for 30 min after the TUNEL assay.

The PBMCs were isolated from the whole blood by the density gradient centrifugation with Ficoll-Paque (Histopaque^®-1077; Sigma-Aldrich, Munich, Germany) according to the protocol described by Uddin et al. [29 (link)]. In brief, whole blood were diluted at the ratio of 1:1 with phosphate buffered saline (PBS) and carefully layered over 8 mL of Histopaque solution previously kept in a 50 mL conical tube. Then the tubes were centrifuged at 1,250x g for 30 min at room temperature. After centrifugation, plasma was aspirated from the upper most layers and kept at −20°C until used. PBMCs preparation was carefully aspirated and treated with RBC lysis buffer (Invitrogen, Darmstadt, Germany) to eliminate erythrocytes. Finally, PBMCs were washed twice with PBS and harvested as pellet.