Collection of brachial lymph nodes (BLNs) for flow cytometry analysis.
BLNs were harvested in 2 mL of RPMI containing 0.26 U/mL Liberase TL and 25 µg/mL DNase I (Sigma Aldrich). Each BLN was flushed using a syringe and incubated 20 min at 37°C. The enzymatic reaction was then stopped with 250 µL of EDTA 100 mM. Cells were homogenized with a 100 µm cell strainer in magnetic-activated cell sorting buffer (Miltenyi Biotec) and counted. Cells were incubated for 15 min at 4°C with Fc Block (BD Biosciences) and stained for 25 minutes at 4°C with fluorochrome-conjugated antibodies: anti-CD11c-PE (clone REA754, Miltenyi Biotec), anti-MHC-II-VioBlue (clone REA813, Miltenyi Biotec), anti-CD11b-PerCP-Vio700 (clone REA592, Miltenyi Biotec), anti-EpCAM-PE-Vio770 (clone caa7-9G8, Miltenyi Biotec), anti-XCR1-APC-Vio700 (clone REA707, Miltenyi Biotec). Additionally, dead cells were excluded with Zombie Aqua (Biolegend) staining. Cells were acquired on MACSquant 10 flow cytometer (Miltenyi Biotec) and data were analyzed using FlowJo software using the gating strategy previously described.9