Anti cd62l apc mel 14

The following monoclonal antibodies were used for murine FACS stainings: From BioLegend (San Diego, CA): anti-CD4 Biotin (GK1.5, 1:400), anti-CD8a Pacific Blue (53-6.7; 1:300), anti-CD11b Pacific Blue (M1/70, 1:300), anti-CD11c Brilliant Violett 421 (N418, 1:400), anti-B220 Pacific Blue (RA3-6B2, 1:300), anti-F4/80 Pacific Blue (BM8, 1:400), anti-CD25 PerCP-Cy5.5 (PC61, 1:200), anti-CD44 PE (IM7, 1:800, 1:3000 for analysis with ≤1,000 cells per well), anti-Ki67 APC (16A8, 1:400); anti-Ki67 Brilliant Violett 605 (16A8, 1:400), anti-CD90.1 PerCP-Cy5.5 (OX-7, 1:500), anti-CD90.2 APC-Cy7 (30-H12, 1:500); from eBioscience (San Diego, CA): anti-CD4 Alexa Fluor 700 (RM4-5; 1:200; 1:600 for analysis with ≤1,000 cells), anti-CD62L APC (MEL-14, 1:400), anti-Foxp3 FITC (FJK-16s, 1:200), polyclonal donkey anti-rabbit IgG PE (1:2000); from BD Biosciences: anti-CD14 V450 (rmC5-3, 1:400). Unspecific binding of antibodies was prevented by incubation of cell suspensions with Fc-Block (BD Pharmingen, 2.4G2, 1:100) for 10 min on ice, followed by flow cytometric staining for 30 min on ice in the dark. Cells were passed through a 40 μm cell strainer (NeoLab) to remove large debris. Enumeration of cells and acquisition were performed by using FACSAriaIII and FACSDiva software (BD version 6.1.3). Single-cell data analyses were performed by the use of the FlowJo software 7.6.1 (Tree Star Inc., OR).