Isolation of Peripheral Blood Mononuclear Cells

Mice were anesthetized with isoflurane and killed by cervical dislocation. The thoracic cavity was opened, and blood was aspirated from the right ventricle with a syringe and transferred to EDTA tubes. Blood was mixed 1:1 with sterile PBS (1X, containing 2 mM EDTA) and layered onto Ficoll-Paque Plus (1.5-fold volume of blood, density = 1.077 g/mL). Density gradient centrifugation was performed for 40 min in a swing out centrifuge at 400 G with breaks turned off. Buffy coat layer containing PBMC was transferred, washed with PBS (1X, 2% FCS, 2 mM EDTA) and spun down for 10 min at 300 G. For platelet depletion, PBMCs were washed with PBS and spun down for 15 min at 200 G. Platelet contamination was evaluated by microscopy and cell-platelet ratios higher than 10 cells per platelet were considered as satisfactory. Cell pellets were resuspended in cell culture medium. Cells were cultured in RPMI-1640 (+10%FCS; +1% Penicillin/Streptomycin; 2 mM L-Glutamine) and further incubation was carried out in an CO₂ incubator at 37°C with 5% CO₂ gas supplement and >90% humidity.