Because of ethical considerations, human LNs were obtained from patients with head and neck cancer undergoing lymphadenectomy as a diagnostic procedure. This project was reviewed and approved by the Institutional Review Board, Chang Gung Medical Foundation (no. 1812210039). All samples were obtained after informed consent. Mononuclear cell suspension was obtained immediately after surgery by mechanically crushing the sample using a scalpel and Frosted Microscope Slides followed by syringing through a 23-gauge needle. All steps were performed on ice. The cell suspension was washed in cold PBS (Sigma-Aldrich) with 1% BSA (Sigma-Aldrich), resuspended at 107 cells/ml, and used for immunostaining.

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