Apc anti mouse ifn γ clone xmg1

Monoclonal antibodies Brilliant Violet 421 anti-mouse CD3 clone 17A2, PE anti-mouse CD4 clone GK1.5, Brilliant Violet 650 anti-mouse CD8 clone 53-6.7, APC anti-mouse IFN-γ clone XMG1.2, PE/Cy7 anti-mouse TNF-α clone MP6-XT22, and Alexa Fluor 488 anti-mouse interleukin-2 (IL-2) clone JES6-5H4 were obtained from BioLegend (San Diego, CA, USA). Intracellular cytokine staining was performed as described previously [42 (link)]. Briefly, 2 × 10⁶ splenocytes were plated into each well and stimulated with 5 μg/mL HA peptide pool or DMSO (negative control) for 16 h. Then, 5 h prior to the end of stimulation, 1 μg/mL brefeldin A (BioLegend) was added to each well. Cells were then washed with FACS buffer (2% FBS in sterile DPBS) and surface staining was performed in the dark on ice for 30 min. Cells were fixed with IC FIX buffer (BioLegend), and permeabilized with 1X permeabilization buffer (BioLegend). Intracellular cytokine staining was then performed in the dark on ice for 40 min. The stained samples were then washed twice with FACS buffer and fixed with 0.5% PFA. For each sample, 1 × 10⁶ events were acquired on a BD LSRII Fortessa, and data were analyzed using Flowjo software V10.

Hybridoma producing anti‐l‐selectin monoclonal antibody Mel‐14 was grown and the antibody purified according to standard procedures. FTY720 was purchased from Sigma‐Aldrich. TLR‐grade Re‐form LPS from E. coli serotype R515 was purchased from Enzo Life Sciences. The antibodies used were as follows: anti‐asialo GM1 (Functional Grade Purified, cat. no. 16‐6507; eBioscience); PE anti‐mouse CD49b (clone DX5, cat. no. 108908; Biolegend); APC anti‐mouse IFN‐γ (clone XMG1.2, cat. no. 505810; Biolegend); FITC anti‐mouse CD3 (clone 17A2, cat. no. 100204; Biolegend); APC anti‐mouse CD11c (clone N418, cat. no. 117310; Biolegend); APC/Cy7 anti‐mouse CD3 (clone 17A2, cat. no. 100222; Biolegend); purified anti‐mouse CD31 (clone MEC 13.3, cat. no. 102502; Biolegend); PE/Cy7 anti‐mouse CD45.2 (clone 104, cat. no. 109830; Biolegend); and anti‐IL‐18 polyclonal antibody (cat. no. sc‐7954‐Y; Santa Cruz Biotechnology).