Cx3cr1 clone sa011f11

Lymphocytes were counted and subjected to viability staining (Fixable Aqua Dead Cell staining, Life Technologies, #L34957) and subsequently to receptor Fc blockade (BD #553142). Staining was performed using the following antibodies: CD4 (clone RM4-4, BD), CD45 (clone 30-F11 eBioscience), CD45.1 (clone A20, BD), CD45.2 (clone 104, BD) CD8a (clone 53-6.7, eBioscience or BD), CD8b (clone YTS156.7.7, BioLegend), CD11b (clone RM2817, Thermo Fisher), Ly6c (clone AL-21, BD), CD3ε (clone 145-2C11, BD), CD19 (clone 1D3, BD), CD90.2 (53-2.1, BD), CD127 (clone A7R34, BD), CX3CR1 (clone SA011F11, BioLegend), CXCR3 (clone CXCR3-173, Biolegend), CD103 (clone 2E7, eBioscience or Biolegend), CD69 (clone H1.2F3, BD), TCR-β (clone H57-597, BD, TCR-γδ (clone eBio-GL3, eBioscience). H2-K^b:SIINFEKL or H2-M3:fMIGWII tetramers were either produced in house or, for the former, obtained thorugh the NIH tetramer core. Samples were fixed (IC Fixation Buffer, eBioscience), washed, resuspended in FACS buffer and acquired with a LSRII flow cytometer (BD) either immediately or on the following day. For intracellular staining, the following antibodies were used: IFN-ϒ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) in Permeabilization buffer (eBioscience).

The following anti-mouse antibodies were used (dilutions are indicated): CD45 (clone 30-F11, 1:100), CD11b (clone M1/70, 1:300), Ly6C (clone HK1.4, 1:300), CD115 (clone AFS98, 1:100), CD64 (clone X54-5/7.1, 1:50), Ly6G (clone 1A8, 1:100), and Tim4 (clone RMT4-54, 1:50), MHCII (clone M4-114.15.2, 1:200), CX₃CR1 (clone SA011F11, 1:100), CD14 (clone M5E2, 1:100), CD16 (clone 3G8, 1:100) all purchased from BioLegend, San Diego, CA, USA. Anti-mouse F4/80 (clone A3-1, 1:50) was purchased from BIORAD. Cells were analyzed with BD FACSCanto™ II (BD Bioscience). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR, United States).