Ficoll hypaque density gradient solution

In brief, heparinized venous blood obtained from healthy volunteers was mixed with a 2% dextran solution (mol. wt. 464,000 daltons; Sigma-Aldrich Chemical Company, St. Louis, MO, USA) at a ratio of four parts blood to one part dextran, and the mixture was incubated at room temperature for 30 min. A leukocyte-enriched supernatant was collected and layered over a Ficoll–Hypaque density gradient solution (specific gravity 1.077; Pharmacia Biotech, Uppsala, Sweden). After centrifugation at 250× g for 25 min, PBMCs were aspirated from the interface. Then PBMCs (1 × 10⁶/mL) were stimulated with 1 μg/mL anti-human CD3 and 1 μg/mL anti-human CD28 (BioLegend, San Diego, CA, USA) plus different concentrations of BDNF (0, 20, or 200 ng/mL) at 37 °C in 5% CO₂ for 24 h. After culture, cells were pelleted by centrifugation at 300× g, and the supernatant was concomitantly collected and stored at −80 °C for the measurement of cytokines.

All participants signed an informed consent under a study protocol approved by the institutional review board of Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (No. B10604013). Heparinized venous blood from healthy individuals was mixed with one-fourth volume of 2% dextran solution (MW 464 kDa; Sigma-Aldrich) and incubated at room temperature for 30 min. Leukocyte-enriched supernatants were collected and layered over a Ficoll-Hypaque density gradient solution (specific gravity 1.077; Pharmacia Biotech, Uppsala, Sweden). After centrifugation, peripheral blood mononuclear cells (PBMCs) were aspirated from the interface. These PBMCs were differentiated to macrophages using Macrophage Generation Media (PromoCell, Heidelberg, Germany) according to the manufacturer’s protocol.

This study was approved by the IRB and Ethical Committee, National Taiwan University Hospital, Taipei, Taiwan (201401102RIN). Informed consent was obtained from each participant. Heparinized venous blood was mixed with one-fourth volume of 2% dextran solution (mol. wt. 464,000 daltons) (Sigma-Aldrich Chemical Company, St. Louis, MO, USA) and incubated at room temperature for 30 min. Leukocyte-enriched supernatant was collected and layered on a Ficoll-Hypaque density gradient solution (specific gravity 1.077) (Pharmacia Biotech, Uppsala, Sweden) followed by centrifugation at 250× g for 25 min. The MNCs were aspirated from the interface. The cell concentration of MNCs was adjusted to 2 × 10⁶/mL in 10% fetal bovine serum in RPMI-1640. The MNC suspension contained 90% lymphocytes, 5%–8% monocytes, and 2%–5% other cell populations confirmed by Giemsa stain with a viability greater than 95%, as confirmed by trypan blue dye exclusion.