Isolation of Mononuclear Cells from Tonsil

The isolation of mononuclear cells from the tonsil was performed as previously described [28 (link)]. Briefly, mononuclear cells were incubated for 15 min at RT with the lymphocyte-specific antibodies, anti-CD3 (clone 8E6), anti-CD8a (PT36A) (both from Washington State University Monoclonal Antibody Centre, Pullman, WA, USA), anti-CD21 (clone BB6-11C9.6, Southern Biotech, Cambridge Bioscience, Cambridge, UK) and anti-IgM (Clone K52 1C3, Bio-Rad) antibodies in PBS with 2% FBS (FACS buffer). After incubation, the cells were washed twice in a FACS buffer and then incubated with anti-mouse IgG microbeads (Miltenyi Biotec) for 15 min at room temperature. The cells were then washed twice in a FACS buffer and resuspended in a FACS buffer supplemented with 2 mM EDTA (MACS buffer). Cells were then applied to LD columns held in a MidiMACS magnet (Miltenyi Biotec). The flow-through, containing the lymphocyte depleted fraction and the two 1 mL washes with MACS buffer were collected in 15 mL tubes. The cells were washed twice with a FACS buffer and resuspended in 10 mL of RPMI-1640 (Life Technologies) supplemented with 10% FBS (Autogen Bioclear, Calne, UK), (cRPMI), before cell counting by volumetric flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec).