Ly6c al 21

Single-cell suspensions from spleens were stained with the Live/Dead Cell Viability assay (Invitrogen), and cell surface markers were stained with the following anti-mouse antibodies at 1:200 dilution per 10⁶ cells: Ly6C (AL-21; BD Biosciences), Ly6G (1A8; BD Biosciences), B220 (RA3-6B2; BioLegend), CD11B (M1/70; BioLegend), TCRb (H57-597; BioLegend), CD11C (N418; BioLegend), and CD45 (30.F11; eBioscience). All samples were analyzed using a FACS Fortessa cytometer (BD Biosciences).

Cells were incubated with CD16/CD32 Fc blocking antibody (2.4G2, 1:100; BD Biosciences, San Jose, CA, USA) for 10 min, then stained with fluorochrome-conjugated antibodies for 30 min on ice. CD11c (N418, 1:200), Ly6G (1A8, 1:300), CD115 (AFS98, 1:300), CD206 (C068C2, 1:400), F4/80 (BM8, 1:300), MHC II (M5/114, 1:300), Siglec-F (E50-2440, 1:300), and 7-AAD Viability Staining Solution were purchased from BioLegend (San Diego, CA, USA). CD11b (M1/70, 1:400), Ly6C (AL-21, 1:200), CD8a (53-6.7, 1:200), CD3 (17A2, 1;300), DAPI solution, and Annexin V were purchased from BD Biosciences. We performed flow cytometry with an LSRFortessa flow cytometer and sorted cells with an FACSAria III cell sorter (both from BD Biosciences). For data analysis, we used FlowJo software version 10.4 (Ashland, OR, USA).

The isolated ileal muscle layer was starved for 30 min in Ca²⁺-free Hank’s solution and then digested for 1 h with continuous stirring at 37 °C in the presence of collagenase (Worthington Type II, Wako), bovine serum albumin (BSA; Sigma-Aldrich Japan), trypsin inhibitor (Sigma-Aldrich Japan), and adenosine triphosphate (ATP; Sigma-Aldrich Japan) in Ca²⁺-free Hank’s solution. Leukocytes were stained with monoclonal antibodies including anti-CD45 (30-F11; 25–0451; eBioecience, San Diego, CA), 7-Aminoactinomysin (7-AAD; 559925; BD Pharmingen Japan, Tokyo, Japan), CD11b (M1/70; 11–0112; eBioscience), Gr-1 (RB6–8C5; 12–5931; eBioscience), and Ly6C (AL-21; 560596; BD Pharmingen Japan). Samples were acquired and evaluated using a FACSVerse (BD Biosciences, Tokyo, Japan). For all samples, approximately 30,000 live 7-AAD-negative cells were analysed for plot generation.

Immunofluorescence was performed on OCT-embedded frozen tissue sections. Sections were blocked with 10% goat serum and 10% BSA in PBS for 1 hour at RT, incubated with primary antibody overnight at 4°C, then washed and incubated with Al-448 or Al-466 goat anti-rat secondary antibody (Invitrogen) at 1∶800 dilution. Immunohistochemistry was performed on formalin-fixed paraffin-embedded tumor sections. Sections were subject to heated antigen unmasking solution for 30 minutes, quenched in 0.3% hydrogen dioxide in PBS for 15 minutes, blocked with 10% goat serum and 10% BSA in PBS for 1 hour at RT, then incubated with primary antibody overnight at 4°C, incubated with biotinylated goat anti-rat secondary antibody at 1∶800 dilution for 30 minutes at RT, and developed using the ABC kit (Vector Labs). Slides were counterstained with hematoxylin. Sections were mounted with Vectorshield and analyzed by fluorescence or light microscopy (Zeiss). Antibodies include CD11b (M1/70, R&D Systems), CD4 (RM4–5, Biolegend), CD8 (53.6–7, R&D Systems), CD49b (DX-5, Biolegend), Foxp3 (FJK-16s, eBioscience), Ly6G (RB6-BC5, eBioscience), Ly6C (AL-21, BD Biosciences), E-Cadherin (MAB7481, R&D Systems), N-cadherin (H-63, Santa Cruz Biotechnology), and phospho-IκBα (Ser 32/36, #9246, Cell Signaling).

Single-cell suspensions prepared from spleens, dissociated tumor cells, or differentiated bone marrow cells were analyzed by flow cytometry. Antibodies including CD4 (RM-4), CD8 (53–67), NK1.1 (pk-136), B220 (RA3-6B2), CD11b (M1/70), CD45.2 (104), Gr1 (RB6-8C5), Ly-6G (1A8), and Ly-6C (AL21) were obtained from BD Biosciences. Data acquisition was performed on a FACS LSRII (BD Biosciences) and analyzed using FlowJo.