Ficoll plaque plus

This research followed the tenets of the Declaration of Helsinki, and protocols for this study were approved by the by the Internal Research Board of Taipei Veterans General Hospital (Board No. 2013-11-012B). All samples were obtained after patients had given informed consent. PBMC was isolated from five patients with dry AMD and two unaffected control using Ficoll-Plaque Plus (Amersham Biosciences) according to the manufacturer's protocol. The characteristics of 5 patients with AMD were summarized in Figure 1C. In brief, one ratio of blood sample was layered on one ratio of Ficoll-Plaque Plus, pellet (400 × g, 30 min at 20°C) and the buffy coat was collected, washed twice with PBS and cultured in DMEM (Sigma) (100 IU/mL of penicillin, 100 μ g/mL of streptomycin [Flowlab] and 10% v/v Fetal Bovine Serum [FBS] [PAA, Austria]). T cells were expanded in freshly prepared AIM-V Medium (Invitrogen) supplemented with pen/strep/glutamine (Invitrogen) plus 300 IU/ml rhIL2 (Peprotech) and 10 ng/ml soluble anti-CD3 antibody (eBioscience, OKT3 clone) (Berger et al., 2003 (link)).

3 mL/kg peripheral blood was obtained from the C57BL/6J mice. Mononuclear cells (MNCs) were isolated by Ficoll-Plaque Plus (Amersham Biosciences) with density gradient centrifugation. MNCs were then rinsed and seeded on 6-well plates coated with human fibronectin (Sigma-Aldrich) and supplemented with EGM-2 (Cambrex). On 7 days of culture, the adherent cells (as early EPCs) were collected for transplantation. The early EPCs were maintained subsequently with acetyl-LDL (10 µg/mL; Molecular Probes, Carlsbad, USA) and isolectin (5 µg/mL; Molecular Probes). The staining of acetyl-LDL and isolectin in EPCs was determined under fluorescence confocal microscopy with the absorption wave lengths of 555 and 495 nm, respectively. EPCs (3 × 10⁵ cells) were harvested for trypsinization 7 days after seeding for transplantation.

Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Plaque Plus density gradient centrifugation (Amersham Biosciences, NJ, USA). Fresh tissues were washed and cut into small fragments, prior to homogenization by tissue disaggregation vessels (BD Medimachine System). The cells in each sample were adjusted to a concentration of 2 × 10⁶ cells/mL, and 0.5 mL cell suspension stimulated with 2μL Leukocyte Activation Cocktail and BD GolgiplugTM (BD Pharmingen, San Diego, CA, USA) for 4 hours.
T cell subsets were phenotyped in isolated PBMCs or tissue cells by flow cytometry (FACSAria flow cytometer, BD Biosciences, NJ, USA) according to manufacturer’s instructions. The cells were labelled with specific monoclonal antibodies; including anti-CD4-PerCP, anti-CD25-PE, anti- Foxp3-FITC, anti-IL-4-PE and anti-IFN-γ-FITC (all from BD Biosciences, NJ, USA). T cell subsets were selected for detailed phenotypic analysis as follow: (1) Th1 cells: IFN-γ⁺IL-4^- CD4⁺ T cells; (2) Th2 cells: IFN-γ^-IL-4⁺ CD4⁺T cells; (3) Treg cells: CD4⁺CD25⁺Foxp3⁺ T cells. A minimum of 10⁶ cells per staining were assessed, with at least 10⁵ events being measured. To measure CCRs in peripheral Treg cells, anti-CCR3, anti-CCR4, anti-CCR5 and anti-CCR8 (R & D Systems, Mineapolis, MN, USA) were used to evaluate corresponding receptor expression in circulating Treg cells of 3 groups.