Anti f4 80 apc cy7

After the intervention, Mφs were collected and incubated with anti-F4/80-APC/Cy7 (BioLegend), anti-CD11b-FITC (BioLegend), anti-CD86-PE (BioLegend), and anti-CD206-APC (BioLegend) antibodies. Flow cytometry was performed on an FACSCalibur (BD Biosciences) and analyzed using FlowJo software. The gating strategy for M2 polarization was as follows: the CD11b⁺- and F4/80⁺-populations were gated first, and then the CD11b⁺ F4/80⁺ population was further gated based on CD206 intensity.

For cell death analysis, cells were stained with Pacific blue-labeled annexin V from Biolegend (San Diego, CA, USA) and propidium iodide (PI) (Sigma-Aldrich) for 15 min in the dark and analyzed by flow cytometry (BD FACSVerse, BD Biosciences). For cell cycle analysis, the cells were fixed with ice-cold 70% ethanol, stained with a solution containing PI, RNase A and Triton X-100 (all Sigma-Aldrich) in PBS for 30 min at 4 °C and then analyzed by flow cytometry (BD FACSVerse analyzer, BD, Allschwil, Switzerland).
For TIL analysis, samples were preincubated with anti-mouse CD16/CD32 (BioLegend) to block Fc receptors and dead cells were excluded by Zombie Aqua staining (Biolegend). The following antibodies were used: anti–CD45-Pblue, anti–CD4-AF700, anti–NKp46-APC, anti–CD11b-PE and anti-F4/80-APC-Cy7 from BioLegend (San Diego, CA) or anti–CD8-PE (BD Pharmingen), and isotype-matched antibodies from Sigma-Aldrich. Data acquisition was done on a BD FACSVerse and data analysis with FlowJo (Tree Star, Stanford, CA, USA).

Immune cells from the colon were isolated according to recent protocol^{63 (link)}. The isolated immune cells were resuspended in FACS buffer (2% FCS in PBS) and were passed through a 70-μm strainer (BD Biosciences). Samples were analysed with following antibodies: anti-CD11b-PE (1:100 dilution, 101208, Biolegend), anti-CD11c-PE-Cy7 (1:100 dilution, 117318, Biolegend), anti-γδTCR-APC (1:100 dilution, 118116, Biolegend), anti-γδTCR-PE-Cy7 (1:100 dilution, 118123, Biolegend), anti-βTCR-FITC (1:100 dilution, 109205, Biolegend), anti-βTCR-PE (1:100 dilution, 553172, BD Biosciences), anti-CD45-V510 (1:100 dilution, 103137, Biolegend), anti-CD45-PE-CF594 (1:200 dilution, 562420, BD Biosciences), anti-CD45-APC (1:200 dilution, 103112, Biolegend), anti-CD90.2-PE-Cy7 (1:200 dilution, 140309, Biolegend), anti-CD3-PE-CF594 (1:200 dilution, 562332, BD Biosciences), anti-F4/80-APC-Cy7 (1:50 dilution, 123119, Biolegend), anti-CD19-FITC (1:100 dilution, 115505, Biolegend), anti-CCR-6-V421 (1:50 dilution, 129817, Biolegend), anti-IL-6R-PE (1:50 dilution, 115805, Biolegend) and LIVE/DEAD Fixable Violet Dead Cell Stain Kit or LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (1:1000 dilution, L34955 and L34957, ThermoFisherScientific). Data were acquired on MACSQuant Analyzer (Miltenyi) and MACSQuant VYB (Miltenyi) and data were analysed with FlowJo software (Treestar).