Anti plzf d 9

Cells were harvested, stained, acquired on a FACSCantoII (Becton Dickinson) and analyzed with FlowJo (Treestar). Dead cells were excluded using the Fixable Viability Dye eFluor506 (eBioscience). Antibodies conjugated to FITC, PE, PerCP Cy5.5, PE Cy7, APC, APC-Cy7 or Pacific Blue were purchased from BD Biosciences, eBioscience, BioLengend and R&D Systems. PE- or APC- conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) were obtained from the tetramer facility of the US National Institutes of Health. In brief, cells were incubated with FC block and stained with antibodies, and then fixed with 2% paraformaldehyde. For PLZF, T-bet, and TCF-1 intracellular staining, cells were permeabilized and stained with anti-PLZF (D-9) (Santa Cruz Biotechnology, Inc.) plus FITC anti-mouse (BD Biosciences), anti-T-bet PerCP-Cy5.5 (eBio4B10), both purchased from eBioscience, and with anti-TCF-1 (C63D9) (Cell Signaling) followed by goat anti-rabbit-Alexa647 (Invitrogen), using the Foxp3 Staining Buffer kit (eBioscience).