Anti nk1.1 apc

Single‐cell spleen suspensions were prepared at 0, 4, 5, 6, 7, 8, 9, 10, 14, 17, 21, 24 and 28 days post‐infection (dpi) as previously described.13 Unless otherwise stated, cell suspensions were re‐suspended in 0.05% FBS BD FACSFlow Sheath Fluid. Cell washings were carried out by centrifugation at 314 g for 7 minutes. Incubations were performed at 4°C for 30 minutes. Non‐specific binding sites were blocked using anti‐CD16/CD32 (Fc γ III/II block—final dilution 1/1000). Afterwards, 5 × 10⁵ cells were incubated with antibody cocktails (dilution of 1/600), using anti‐B220‐FITC, anti‐CD1d‐PE, anti‐CD138‐PE/CY7, anti‐CD93‐APC, anti‐CD4‐FITC, anti‐CD8a‐PE, anti‐TCR β chain‐APC, anti‐Ly6G‐AlexaFluor488, anti‐Ly6C‐PE, anti‐CD11b‐APC, anti‐NK1.1‐APC and anti‐Ter119‐PE (BioLegend, San Diego, CA, USA), 1 µg of 7‐amino‐actinomycin D (7AAD) to exclude nonviable cells, and finally analysed using a BD Accuri™ C6 Plus flow cytometer.

Single cell suspensions of splenocytes or peripheral blood were prepared using the standard techniques. Fc receptors were blocked by using 2.4G2 mAb prior to surface staining with the indicated antibodies. The negative selection of NK cells was determined by staining with cell surface monoclonal antibodies (mAbs), including anti-CD19, anti-CD4, anti-CD8a, anti-CD5, anti-Gr1, and anti-Ter-119 (Miltenyi Biotech). The surface phenotype of NK cells was determined by staining with mAbs, including anti-CD3-PE and anti-NK1.1-APC (Biolegend, San Diego, CA). Intracellular IFNγ was measured by pacific blue-conjugated anti-IFNγ (Biolegend) as described previously [47 (link)]. Dead cells were excluded by propidium iodine (PI) staining and live cells were gated on PI-negative cells. The NK cells of donors or recipients were distinguished by using anti-CD45.1 mAb (A20, BD Biosciences, San Jose, CA), FITC-34-2-12 (H-2D^d mAb, BD Biosciences), or Bio-KH95 (anti-H-2D^b mAb, BD Biosciences). Data acquisition was performed with FACS Calibur flow cytometer (BD Pharmingen, San Jose, CA) using a CellQuest software (BD Biosciences). Data analysis was performed using a FlowJo software [48 (link)].