Isolation of Peripheral Blood and Lymph Node Cells

Heparinized peripheral blood was obtained from all patients and healthy controls. All heparinized blood samples were diluted with an equal volume of 0.9% saline, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway) density gradient centrifugation [23 (link)]. Following centrifugation at 800 × g for 20 min at room temperature, PBMCs were collected and washed twice with 0.9% saline. FNA samples were taken from palpable lymph nodes, and lymph nodes cells (LNCs) were suspended in 500 μl of saline. LNCs were centrifuged for 15 min at 1,500 rpm, and the supernatant was discarded. Cells were resuspended in 1 ml of VersaLyse (Beckman Coulter, Marseille Cedex, France) and incubated for 5 min at room temperature in the dark. Following centrifugation, LNCs were washed and resuspended in 0.9% saline.