Lymphocyte separation media

To perform the chemotaxis assay, the healthy donor's peripheral blood mononuclear cells (PBMCs) were separated by the gradient centrifugation method using lymphocyte separation media (Biosera, France). Like the migration assay, PBMCs were seeded in the upper chamber and cultured for 6 hours. After that, the lower chamber medium was collected for counting. The cell number was determined by adding counting beads by flow cytometry. 10000 particles of beads were mixed with each group of medium containing migrating PBMC. The flow cytometry was performed by LSRFortessa (BD Biosciences, USA).

Peripheral blood samples were obtained from healthy donors. Informed consent was provided according to the Declaration of Helsinki. The PBMCs were isolated by density gradient centrifugation while using Lymphocyte Separation Media (Biosera, Monza, Italy). The DCs were prepared, as previously described [29 (link)], with minor modifications. Briefly, monocyte purification was obtained by positive selection, while using anti-CD14 conjugated magnetic microbeads (Miltenyi Biotec, Bologna, Italy). Cell purity, as assessed by flow cytometry (FACS), was always ≥ 95%. Freshly purified monocytes were cultured for five days at the density of 350,000 cells/ml in complete medium (RPMI 1640, 10% endotoxin-free FBS, 2 mM L-Glutamine, and 1% Penicillin-Streptomycin) supplemented with 20 ng/mL of recombinant human IL-4 and 50 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) both purchased from Miltenyi Biotec to obtain immature DCs (immDCs).

Ten ml of venous blood were collected in vials containing ethylenediaminetetraacetic acid (EDTA). This was performed at the beginning of therapy (day 1 of RT) and one week after the last fraction of RT.
Plasma and peripheral blood mononuclear cells (PBMCs) were isolated after density gradient centrifugation using a 1.077 g/mL synthetic epichlorohydrin sucrose polymer Lymphosep [Lymphocyte Separation Media-500 mL; density 1077 g/mL; Cat no: LM-T1702/500; Biosera, Cholet, France], at a 1:1 ratio of the total blood volume. Centrifugation of blood samples was performed at 1500 rpm (30 min, 25 °C). The plasma layer was transferred in sterile Eppendorf tubes and was gradually frozen at −20 °C and subsequently at −80 °C. The PBMC layer was also collected for further studies and stored at −20 °C.