Ly 6c efluor 450

For surface and intracellular staining, the monoclonal antibodies listed were used: CD4 (PerCP) (RM4-5); CD192 (CCR2) (Alexa 647) (SA203G11) all from BioLegend (Biozol); CD8 (53-6.7); IL-4 (PE-Cy7) (11B11); IL-5 (PE) (TRFK5); IL-13 (Alexa 488) (eBio13A); IFN-γ (eFluor 450) (XMG1.2); CD154 (PE) (MR1); Foxp3 Alexa 488 (FJK-16s); GATA3 (eFluor 660) (TWAJ); Dead Cell Exclusion Marker (DCE) (efluor 780); DCE (efluor 506); Siglec F (PE) (E50-2440); T-bet (PE) (eBio4B10); IL-13 (eFluor 660) (eBio13A); CD11b (PE) (M1/70); F4/80 (PerCP-Cy5.5) (BM8); Ly-6G (Gr-1) (PE-Cy7) (RB6-8c5); Ly-6C (eFluor 450) (HK1.4); TNF-α (Alexa488) (MP6-XT22) all from eBioscience, San Diego, CA, USA.
For intracellular staining of cytokines and transcription factors cells were fixed and permeabilized using the fix/perm buffer kit (eBioscience, San Diego, CA, USA). FACSCantoII flow cytometer and FACSAriaIII sorter (both BD Bioscience, Heidelberg, Germany) were used for cell analysis. FlowJo software 10.2 was used for final analysis (Tree star Inc., Ashland, OR, USA).

Single cell suspensions from mouse experiments were stained with Live/dead Aqua (Molecular Probes, Inc.), CD4-PerCP, CD8-Pacific Blue, CD69-BV605, CD62E-PE, CD62E-BV421, CD62P-Alexa647, CD162-AlexaFlour647, HECA454-PE (BD Bioscience), CD25-APC, CD31-BV605, CD105-Pacblue, I/A-I/E-BV421, CD45-APCCy7, EpCAM-APC-Cy7, Podoplanin-PE, CD31-PECy7, EpCAM-FITC, CD64-BV711 (Biolegend), CD11c-PECy5.5 (Invitrogen) CXCR3-PECy7, CD3-Alexa700 and Ly6C-efluor450 (eBioscience). For subsequent detection of CXCL10 mRNA Primeflow^® RNA Assay (eBioscience) was used accordingly to manufacture protocol. Samples were acquired on an LSR-II flow cytometer (BD Biosciences) and analysed using FlowJo software (Tree Star Inc.).
Single cell suspension isolated from human tumors and unaffected colon tissue were stained with Live/dead Aqua (Molecular Probes, Inc.), CD31-Alexa700, (Biolegend), CD4-PerCP, CD8-BV711, CD105-APC, CD14-Alexa700, CD19-APCH7 and CD19-PE-CF594 (BD bioscience) followed by permeabilization with Fix & Perm kit (ADG Bio research GMBH) and staining with CXCL9-FITC (R&D) and CXCL10-PE (Biolegend), flow cytometry analyses were performed as above.

Aliquots of up to 3 × 10⁶ cells were blocked using an anti-CD16/CD32 blocking antibody (BioLegend, San Diego, CA) and labelled with fluorescently conjugated antibodies in the dark as described previously (Thomson et al. 2018). The following antibodies were purchased from BioLegend: CD4-AF488 (GK1.5), CD8α-PE/Cy7 (53-6.7), CD11b-PE (M1/70), CD44-BV421 (IM7), CD45-BV510 (30-F11), CD45-PE/Cy7 (30-F11), CD62L-PE (MEL-14), CD64-APC (X54-5/7.1), F4/80-FITC (BM8), Ly6G-AF700 (1A8), MHCII-BV510 (M5/114.15.2), NK1.1-BV605 (PK136) and TCRβ-PerCP/Cy5.5 (H57-597). The following were purchased from eBioscience (San Diego, CA): CD11b-AF700 (M1/70) and Ly6C-eFluor 450 (HK1.4). Dead cells were excluded from analysis using Fixable Viability Dye eFluor 780 (eBioscience, San Diego, CA). Cells were analysed using an LSRII (BD Biosciences, San Jose, CA). Data were analysed using FlowJo v10 (Tree Star, Ashland, OR).