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