Anti mouse ly6g

The ear dermis/ ear dLN/ lymph nodes/ spleen was removed from animals and single cell suspension was prepared. For surface staining, cells were blocked at 4°C with rat anti-mouse CD16/32 (5 μg/ml) from BD Pharmingen for 20 min. Cells were then stained with anti-mouse Ly6G, anti-mouse CD11b, anti-mouse CD3, anti-mouse CD4, anti-mouse CD44, anti-mouse CCR7, anti-mouse PD-1 (eBioscience) for 30 min (each with 1:200 dilution; at 4°C). The cells were then stained with Live/Dead fixable aqua (Invitrogen/Molecular Probes) to stain dead cells. Cells were washed twice with wash buffer and fixed with a Cytofix/Cytoperm kit (BD Bioscience) for 20 min at room temperature. Intracellular staining was performed with anti-mouse IFN-γ, anti-mouse IL-2, anti-mouse IL-10 (eBioscience) (each with 1:300 dilution; at 4°C). Cells were washed twice with permeabilization buffer 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. 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 and CD8 T cells were identified as CD3⁺ lymphocytes uniquely expressing either CD4 or CD8.