Ki 67 af488

Single-cell suspensions were stained with the appropriate monoclonal antibody in PBS containing 2% FCS. When necessary, intracellular staining was performed by use of the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions. Fortessa, FACS Verse, and ARIAIII (BD Biosciences) were used for cell sorting and analysis. Antibodies specific for NK1.1 (PK136; 1:100), CD19 (1D3; 1:400), CD3 (17A2; 1:400 or REA641; Miltenyi Biotec; 1:150); CD122 (TM-β1; 1:200), NKp46 (29A1.4; 1:100), KLRG1 (2F1; 1:200), CD27 (SB/199; 1:200), CD11b (M1/70; 1:200), IL-7R (A7R34; eBioscience; 1:200) CD49b (DX5; 1:100), CD49a (Ha31/8; 1:200) Ly49H (3D10; 1:200) Ly49D (4E5; 1:200), NKG2D (C4; 1:200), NKG2A/C/E (20d5; 1:200), Ly49C/I (5e6; 1:100), CD107a (104B; 1:100), and IFN-γ (XMG1.2; 1:100), DNAM-1 (10E5; 1:200); Ki-67 (AF488; 1:50) were from BD Pharmingen unless stated otherwise.

Flow cytometry was performed using a BD FACSCantoII™ flow cytometer and analysis run with FACSDiva 6.1.1 software (Becton–Dickinson). Cells were stained with anti-mouse CD45-PB (#103125), CD11b-PE/Cy5 (#101209), F4/80-APC (#123115), F4/80-FITC (#123107), CD206-FITC (#141703), CD80-PE (#104707), SiglecF1-PE (#142403), Gr1-APC/Cy7 (#108423), CD3-APC (#100235), CD4-PE (#100407), CD8-PE/Cy5 (#100709) (BioLegend, San Diego, CA, USA) for 20 min at 4 °C, washed and resuspended in FACS Buffer (PBS, 5 mM EDTA and 0.2% BSA).
Intracellular staining for Ki67-AF488 (#561165, BD Biosciences) was performed by adding 3 ml cold 70% ethanol to the cell pellet and incubating at − 20 °C for 1 h, followed by staining with the antibody at RT for 30 min.