Isolation and Culture of PBMCs from Blood

PBMCs were isolated using combined dextran–ficoll isolation as described previously [34 (link),35 (link)]. In brief, blood was diluted with Dulbecco’s phosphate-buffered saline (DPBS, Gibco™, ThermoFisher Scientific, Waltham, MA, USA) containing 2 mM ethylenediaminetetraacetic acid (EDTA, Carl Roth, Karlsruhe, Germany) 1:1, mixed with 3% dextran (Carl Roth) in a ratio of 1:0.4, and centrifuged at 50× g for 20 min. Thereafter, the upper phase was collected, layered onto an equal volume of Histopaque^®-1077 (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged at 900× g for 30 min. The PBMCs at the interphase were collected, washed twice with DPBS/EDTA in a new 50 mL tube, and centrifuged at 400× g for 10 min. Isolated PBMCs were adjusted to 5×10⁶ cells/mL living cells using Tali™ image-based cytometer (ThermoFisher Scientific) with propidium iodide (PI, ThermoFisher Scientific) as a viability marker. PBMCs were cultured in RPMI 1640 medium (Gibco™, ThermoFisher Scientific) with 10% chicken serum (Gibco™, ThermoFisher Scientific) and 1% penicillin (10,000 U/mL)-streptomycin (10,000 µg/mL) (Pen/Strep, Gibco™, ThermoFisher Scientific) at 41 °C with 5% CO₂. The next day, further experiments were performed.