Flow cytometry

The ear dLNs were removed from animals, and single-cell suspension was prepared. For surface staining, cells were blocked via using rat anti-mouse CD16/32 (Clone: 2.4G2, BD Biosciences, Cat: 553141) at 4°C (5 mg/ml) from BD Pharmingen for 20 min. Cells were then stained with anti-mouse Ly6G (Clone: 1A8-Ly6g, Thermo Fisher Scientific, Cat: 25-9668-82), anti-mouse CD11b (Clone: M1/70, Thermo Fisher Scientific, Cat: 56-0112-82), anti-mouse Ly6C (Clone: HK1.4, Biolegend, Cat: 128006), anti-mouse CD3 (Clone: eBio500A2 (500A2), Thermo Fisher Scientific, Cat: 56-0033-82), anti-mouse CD4 (Clone: RM4-5, Bio legend, Cat: 100552), anti-mouse CD44 (Clone: IM7, Thermo Fisher Scientific, Cat: 48-0441-82), anti-mouse Cd11c (Clone: N418, Thermo Fisher Scientific, Cat: 48-0114-80), anti-mouse Ly6G/Ly6C(Gr-1) (Clone: RB6-8C5, Biolegend, Cat: 108416), anti-mouse CD80 (Clone 16-10A1, BD), anti-mouse MHCII Class II (I-A/I-E) (Clone: M5/114.15.2, Thermo Fisher Scientific, Cat: 47-5321-82), anti-mouse CD40 (Clone: 1C10, Thermo Fisher Scientific, Cat: 17-0401-82), anti-mouse CD205 (Clone: 205yekta, Thermo Fisher Scientific, Cat:25-2051-42), anti-mouse CD8a Antibody (Clone: 53–6.7, Bio legend, Cat: 100711) (each with 1:100 dilution; at 4°C). For MPO staining, cells were stained with MPO Polyclonal Antibody (Clone: bs-4943R, Thermo Fisher Scientific, Cat: BS-4943R) at 1:50 dilution in blocking buffer and incubated for 30 min at room temperature, washed twice with 2%BSA in PBS, followed by secondary antibody incubation for 40 min at room temperature. In order to stain the dead cells for all the flow experiments, the samples were stained with Live/Dead Fixable Aqua (Thermo Fisher Scientific, Cat: {"type":"entrez-nucleotide","attrs":{"text":"L34957","term_id":"522200","term_text":"L34957"}}L34957).

Cells were then washed twice with wash buffer [1x PBS, 2–5% (v/v) FBS (or BSA) 2 mM EDTA] followed by fixation with a Fixation/Permeabilization Solution Kit (BD Bioscience, Cat: 554714) for 20 min at room temperature and acquired on an LSR II (BD Biosciences) equipped with 407-, 488-, 532-, and 633- nm laser lines using FACS Diva 6.1.2 software. Acquisitions of a million events were performed. Data were analyzed with FlowJo software version 9.7.5 (Tree Star). For analysis, first doublets were removed using width parameter; dead cells were excluded based on staining with the Live/Dead Aqua dye. Lymphocytes were identified according to their light-scattering properties. CD4 T cells were identified as CD3⁺ lymphocytes uniquely expressing CD4.