Ficoll paque plus

Blood samples were collected in the morning. PBMCs were isolated using standard Ficoll-Paque Plus (TBD science, China) following the manufacturer’s instructions. The collected cells were used for RNA extractions. Serum samples were stored at −80 °C until the cytokines were determined.

Following shedding of the first 2 ml of peripheral blood to reduce contamination by epithelial cells from the skin, 10 ml peripheral blood was collected in an EDTA-treated vacuum tube from each patient or control subject. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by Ficoll density gradient centrifugation (Ficoll-Paque Plus; TBDscience, Tianjin, China) using standard procedures and frozen at −80°C until RNA extraction.

Fasting venous blood (3 ml per subject) was obtained from the CHD patients and HCs. Blood samples were collected and analysed within 3 h. PBMCs were isolated under sterile conditions via Ficoll-Paque Plus (TBD Science) density gradient centrifugation. The collected cells were stored at −80°C before RNA extraction or before they were cultured. The serum samples were also stored at −80°C before the detection of the cytokines.

Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from blood samples from breast cancer patients or healthy donors from the Guangzhou Blood Center. Briefly, blood samples diluted 1:1 with PBS were gently layered over Ficoll-Paque PLUS (TBDscience, #LTS1077). After centrifugation at 450 × g for 20 minutes with the brake off (Beckman, X-15R), mononuclear cells were collected from the interface, washed 3 times with PBS and resuspended in DMEM. After 20 minutes of incubation at 37°C, the nonadherent cells (periphery blood lymphocytes) were removed to acquire monocytes. Monocyte-derived dendritic cells (mo-DC) were generated by culturing monocytes with 20 ng/mL IL4 (PeproTech, # 200-04-20) and 50 ng/mL GM-CSF (PeproTech, # 300-03-20) for 6 days. CD8⁺ T cells and natural killer (NK) cells were isolated by magnetic-activated cell sorting using direct CD8 and CD56 Isolation Kits (Miltenyi Biotec, #130-094-156 and 130-050-401) according to the manufacturer's instructions. CD8⁺ T cells and NK cells were cultured in RPMI-1640 medium supplemented with 25 mmol/L HEPES (Thermo, # 15630080), 4 mmol/L L-glutamine (Thermo, # A2916801), 25 μmol/L 2-mercaptoethanol (Thermo, # 21985023), and 10% FBS. Media were replaced every 2 days.