Ammonium chloride buffer

All studies were approved by the Institutional Review Board of Washington University in St Louis. Written consent was obtained from all participants. Eight participants who had not been vaccinated against influenza for at least three years were enrolled, including 1 female and 7 males, aged 26–40 years old. PBMCs were isolated using Vacutainer CPT tubes (BD), the remaining red blood cells were lysed with ammonium chloride lysis buffer (Lonza), and cells were immediately used or cryopreserved in 10% dimethylsulfoxide in FBS. Ultrasound-guided FNA of axillary lymph nodes was performed by a qualified physician’s assistant under the supervision of a radiologist. Lymph node dimensions and cortical thickness were measured before each FNA. For each FNA sample, 6 passes were made using 25-gauge needles, each of which was flushed with 3 ml of RPMI 1640 supplemented with 10% FBS and 100 U ml−1 penicillin/streptomycin, followed by three 1-ml rinses. Red blood cells were lysed with ammonium chloride buffer (Lonza), washed twice with PBS supplemented with 2% FBS and 2 mM EDTA, and immediately used or cryopreserved in 10% DMSO in FBS. Participants reported no adverse effects of phlebotomy, serial FNA, or vaccination. No statistical methods were used to predetermine sample size. Investigators were not blinded to experiments and outcome assessment.