Cd3 percp cy5.5 17a2

0.5–1 x 10⁶ ileum and colon cells from each mouse were stained in FACS buffer for 30 min at 4° C with the following antibodies: CD3-PerCP-Cy5.5 (17A2, Biolegend), CD4-FITC (RM4-4, eBioscience), CD8-BV510 (53–6.7, Biolegend), CD69-PE-Cy7 (h1.2F3, eBioscience), CD103-PE-Dazzle-594 (2E7, Biolegend). Mesenteric lymph node cells were additionally stained with CD44-AlexaFluor-700 (IM7, eBioscience), and CD62L-APC-eFluor780 (MEL-14, eBioscience). Cells were then washed with FACS buffer and fixed in 1% paraformaldehyde before being acquired on a BD LSRii flow cytometer. Colon samples that had less than 1,000 T cells were excluded. Plasma samples were thawed and diluted 1:10 before being assayed for sCD14 by ELISA (R&D Systems) according to the manufacturer’s instructions. Ileum and colon tissues cultured for 24 h at 37° C, 5% CO₂ in complete RPMI were digested with CellLytic MT solution with proteinase inhibitor (SIGMA), and assayed for myeloperoxidase concentrations by ELISA (R&D Systems). ELISA plates were read using a Vmax Kinetic plate reader (Molecular Devices). Tissue sections from the distal end of the ileum and proximal end of the colon were processed for histology and H&E stained. Scoring [34 (link)] was performed by a pathologist blinded to experimental conditions.

Single-cell suspensions from mouse subcutaneous tumors were prepared as described above, counted, and resuspended in PBS at a concentration of 1×10⁷ live cells/mL. One hundred microliters of each sample was plated for cell staining. Surface staining was performed at room temperature for 30 min, and intracellular staining was performed using a Foxp3-transcription factor staining kit (eBioscience). Live/dead cell discrimination was performed using Fixable Viability Stain 520 (BD Biosciences). Anti-mouse antibodies against the following antigens were used for flow cytometry: CD3 PerCP-CY5.5 (17A2, BioLegend, 1:100), CD45 PerCP (30-F11, BioLegend, 1:200), NK1.1 APC (PK136, BD Biosciences, 1:50), Foxp3 PE (MF23, BD Biosciences, 1:100), Ly6G PE (1A8, BioLegend, 1:100), Ly6C APC (HK1.4, BioLegend, 1:100), CD11b BV421 (M1/70, BioLegend, 1:100), CD11b PerCP-CY5.5 (HL3, BioLegend, 1:100), F4/80 APC (T45–2342, BD Biosciences, 1:50), CD25APC (3C7, BioLegend, 1:100), CD4 APC (GK1.5, BioLegend, 1:100), and CD8a PE (53-6.7; BioLegend, 1:100). All flow cytometry analyses were performed using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).