Multiparameter Flow Cytometry Analysis

Flow cytometry was performed as previously described [9 (link)]. The following Abs were used: BV785-CD3ε (Clone 145-2C11), BV711-CD8α (Clone 53–6.7), APC/Cy7-CD11b (Clone M1/70), BV-421-Gr-1 (Clone RB6-8C5), PE/Cy7-CD11c (Clone N418), FITC-CD45 (Clone I3/2.3), PerCP/Cy5.5-IA^b (Clone AF6-120.1), BV605-NK1.1 (PK136) were from Biolegend. BUV395-CD4 (Clone GK1.5) and BUV395-CD19 (Clone 1D3) were from BD Biosciences. To obtain single-cell suspensions, LNs were first incubated in Liberase TM (1.67 Wünsch units/mL) (Sigma) in PBS with 25 mM HEPES for 30 min at 37°C before adding PBS with 25 mM HEPES + 10% FBS to halt the digestion process, followed by mechanical disruption of the tissue through a 70-μm filter. Cells were washed once with FACS buffer (PBS with 1% BSA + 0.1% sodium azide) before surface staining. For analysis, samples were acquired using either a BD LSR II flow cytometer or a BD Fortessa flow cytometer (BD Biosciences), and data were analyzed with FlowJo software (TreeStar). For cell sorting, samples were acquired with a BD FACS Aria III sorter (BD Bioscience).