Anti mouse cd16 cd32 mab 93

Lungs were minced and digested with Liberase-TM (Roche, Tokyo, Japan) and DNase (Roche, Tokyo, Japan) for 1 h at 37 °C. After filtration through a nylon mesh (40 μm cell strainer; Greiner, Tokyo, Japan), single lung cells were collected. The cells were incubated with anti-mouse CD16/CD32 mAb (93; eBioscience, San Diego, CA) for FcR blocking for 15 min on ice, and then incubated with each of the following: PE-conjugated anti-mouse CD3ε mAb (145-2C11, BioLegend, San Diego, CA), PE-conjugated anti-mouse Gr-1 mAb (RB6-8C5; eBioscience), PE-conjugated anti-mouse CD19 m Ab (6D; BioLegend), PE-conjugated anti-mouse NK1.1 mAb (PK136; BD Biosciences, San Jose, CA), PE-conjugated anti-mouse CD11b mAb (M1/70; BioLegend), PE-conjugated anti-mouse Ter-119 mAb (TER-119; BioLegend), PE/Cy7-conjugated anti-mouse CD25 mAb (PC61; BioLegend), Brilliant Violet 421-conjugated anti-mouse CD127 mAb (A7R34; BioLegend), Brilliant Violet 510-conjugated anti-mouse CD45 mAb (30-F11; BioLegend), and FITC-conjugated anti-Mouse ST2 mAb (DJ8, MD Bioscience, Zurich, Switzerland). After washing, the cells were suspended in HBSS supplemented with 2% FCS and 7-aminoactinomycin D. The proportions of CD127⁺ CD25⁺ and ST2⁺ CD25⁺ ILC2s among 7-aminoactinomycin D-negative, lineage marker-negative CD45⁺ cells were analyzed on a MACSQuant (Miltenyi Biotec) with FlowJo software (Tree Star).

Fluorescein isothiocyanate (FITC)-conjugated and/or phycoerythrin (PE)-conjugated anti-mouse CD4 (RM4-5; eBioscience, Inc), anti-mouse CD8α mAb (53-6.7; BD Bioscience), anti-mouse/rat Foxp3 mAb (FJK-16s; eBioscience, Inc), anti-mouse IFN-γ mAb (XMG1.2; eBioscience, Inc), anti-mouse F4/80 mAb (BM8; BioLegend), and anti-rat IgG2b monoclonal antibodies (mAbs) (MRG2b-85; BioLegend) as well as a FITC-conjugated rabbit anti-HSV-1 polyclonal antibody (Dako) were used in flow cytometric analysis and immunohistochemstry. An anti-mouse CD16/CD32 mAb (93; eBioscience, Inc) was used for Fc-blocking in all experiments. For in vivo administration, anti-mouse GITR mAb (DTA-1, rat IgG2b) and anti-mouse CD8α mAb were purified by protein G affinity column chromatography of ascites from BALB/c nude mice intraperitoneally inoculated with a 53-6.7 hybridoma. Purifed rat serum IgG (Sigma) was used as the control antibody for all experiments with DTA-1. The Fab portion of DTA-1 was prepared by using a Pierce Fab Preparation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.