Ficoll plaque plus

RAW 264.7 cells, a murine macrophage-like cell line, and DH82 cells, a canine macrophage-like cell line, were purchased from the Korean Cell Line Bank (Seoul, Korea). RAW 264.7 and DH82 cells were seeded in 6-well plates (1 × 10⁶ cells/well) in triplicate and incubated for 24 h. After adherence to the plates was confirmed, the RAW 264.7 and DH82 cells were treated with LPS (200 ng/mL; Sigma-Aldrich) or control for 24 h. Similarly, with the consent of the owners, 10 mL of blood was obtained from each of 3 dogs and canine peripheral blood mononuclear cells (cPBMCs) were obtained using ficoll-plaque PLUS (Sigma-Aldrich) according to the manufacturer’s instructions. cPBMCs were seeded in 6-well plates (1 × 10⁶ cells/well) in triplicate, incubated for 24 h, and exposed to Con A (5 μg/mL) or control for 24 h. Next, the medium was removed and replaced with media containing EVs (50 μg/well) derived from naive (unstimulated) or primed (stimulated) cASCs. Next, the cells were incubated for 48 h and then harvested for RNA extraction and immunofluorescence analysis.

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)).