Nk1.1 apc pk136

Spleens were processed with frosted glass slides and filtered (70 µm) to create single-cell suspensions. Red blood cells were lysed with ammonium-chloride-potassium lysis buffer for 5 min on ice, and remaining cells were washed with PBS before staining. 10⁶ cells per sample were stained with Fixable Live/Dead Near Infrared (#L34975; Thermo Fisher) for dead cell exclusion. Cells were incubated with Fc block (2.4G2; BD Biosciences) for 5 min on ice before staining with the following antibodies at appropriate concentrations for 30 min on ice: CD4-PerCP-Cy5.5 (RM4-5; eBioscience), CD8a-PE-Cy7 (53-6.7; BioLegend), CD3e-BV605 (145-2C11; BioLegend), NK1.1-APC (PK136; eBioscience), CD19-BV711 (6D5; BioLegend), Ly6G-BV421 (1A8; BD Biosciences), Ly6C-PE (HK1.4; eBioscience), and CD11b-AF488 (M1/70; eBioscience). Samples were washed once before data acquisition on an LSR Fortessa (BD Biosciences) and analysis using FlowJo (v10.5.0; TreeStar).

Blood was collected from tail veins in the presence of 5 mM EDTA, incubated with BD Fc Block for 10 min, and then stained with mAbs (BD Bioscience unless indicated otherwise) specific to CD45-APC-Cy7 (clone 30-F11), Ly6G-eFluor450 (1A8-Ly6g, eBioscience), CD11b-PerCP-Cy5.5 (M1/70), Ly6C-FITC (AL-21), NK1.1-APC (PK136), CD3-APC (145-2C11), CD19-APC (1D3), and TER119-APC (TER-119, eBioscience) for 20 min at room temperature. The samples were then fixed and lysed using BD FACS Lysing solution (BD Bioscience). After fixation, cells were washed and resuspended in 2% FBS/PBS. AccuCheck counting beads (PCB100, Invitrogen, Grand Island, NY, USA) were used for quantification of absolute cell numbers. Multiparameter analysis was performed on a FACSSymphony A3 (BD Bioscience) using the FACSDiva software and data were analyzed using the FlowJo software (version 10, Tree Star Inc., Ashland, OR, USA) as described previously [10 (link)].