Anti fasl mfl3

Single cell suspension from the mouse thymus and spleen was prepared as previously described^{18 (link), 21 (link)}. The cells were surface-stained with various combinations of the following antibodies: anti-CD4 (M1/69), anti-CD8α (53-6.7), anti-TCRβ (H57–597), anti-CD69 (H1.2F3), anti-CD44 (IM7), anti-CD24 (M1/69), anti-CD62L (MEL-14), anti-CD40L (MR1) and CD45.2 (104) (all from eBiosciences). Anti-IL-2Rβ (TM-β1) and anti-FasL (MFL3) were from BD Biosciences. Anti-granzyme B (GB/2) was from Thermo Fisher Scientific. The Vβ TCR Screening Panel (BD Biosciences) was used to survey the Vβ TCR repertoire. For intranuclear staining, the surface-stained cells were fixed and permeabilized with Foxp3/transcription factor staining buffer set (eBiosciences), and stained with anti-Foxp3 (eBiosciences, FJK-16s), anti-Rorγt (eBiosciences, B2D), or anti-Tcf1 (C63D9, Cell Signaling Technologies). The stained cells were analyzed on FACSVerse (BD Biosciences). All antibodies were used at 1:50 to 1:100 dilution, and validated for mouse and flow cytometry as indicated by the data sheets from the manufacturers. The data were processed using FlowJo software (Version X, TreeStar). For cell sorting, surface-stained thymocytes or splenocytes were sorted on FACSAria (BD Biosciences) from Tcf7^−/−Lef1^−/− or control mice for RNA-Seq analysis and gene expression validation.

Lymph node cells were stained with the following monoclonal Abs: rat anti-mouse CD8α (53-6.7), hamster anti-mouse CD11c (HL3) or rat anti-mouse CD45R (RA-6B2) (BD Bioscience, San Jose, CA). The anti-FasL (CD178) (MFL3) was purchased from eBioscience (San Diego, CA). For FasL staining, the cells were stained as previously described (27 (link)). Briefly, the cells were blocked with 1:100 rat serum, 1:100 hamster serum and 1:400 free streptavidin (Molecular Probe, Eugene, OR) on ice for 25 min. Cell were then washed twice and stained with biotin conjugated anti-FasL (MFL3) follow by streptavidin PE (BD Bioscience). All flow cytometry data were acquired by BD FACS Calibur or LSRII. The data were analyzed using Flowjo software (TreeStar, Ashland, OR).