Rat igg1 isotype control

Typically, 1–1.5 million CMFDA- or DR-labeled LPS mature DCs (ratio 1:1) were injected in INF or CTR mouse ears or footpads. Auricular and popliteal LNs were harvested at different time points, passaged through a 40-µm cell strainer and stained with BV421 rat anti-mouse MHC-II (BioLegend) and Pe/Cy7 hamster anti-mouse CD11c (BioLegend). Cell suspensions were acquired on a CytoFlex S and the total number of migratory labeled (CMFDA or DR) DCs analyzed in FlowJo. For VCAM-1 blocking experiments in CHS inflammation, 0.5 mg of VCAM-1 blocking antibody was administered i.p. after oxazolone challenge and 8 h before the adoptive transfer. 24 h after challenge, 1:1 ratios of LPS mature tln1^−/− and WT DCs were injected together with 25 µg VCAM-1 blocking antibody (6C7.1; Engelhardt et al., 1998 (link)) or rat IgG1 isotype control (BioXCell) in INF ears and footpads. For integrin α4 blocking experiments, 1:1 ratios of LPS mature tln1^−/− and WT DCs were coinjected with 10 µg integrin α4 blocking antibody (PS/2; Miyake et al., 1991 (link); BioXCell) or rat IgG2b isotype control (BioXCell) in either INF ears or footpads. 14 h (footpads) and 17 h (ear skin) later, dLNs were harvested and processed as described above. The whole LN was acquired on a Cytoflex S, and the total number of migratory labeled (CMFDA or DR) DCs analyzed in FlowJo.

250 μg blocking anti-TNF-α antibody (clone XT3.11) or 250 μg rat IgG1 isotype control (anti-horseradish peroxidase; clone HRPN) (both BioXcell) was injected intraperitoneally into each mouse 3.5 hours prior to administration of LPS or PBS. For experiments analyzed 48 hours after LPS administration, mice were injected a second time with 250 μg anti-TNFα antibody or isotype control antibody 24 hours after LPS administration.