Recombinant il 13

The human colorectal cancer cell line, HT‐29 (a gift from Professor Phil Darcy), was grown to near confluence, trypsinized, and re‐seeded at 100 000 cells per well in a 96‐well U‐bottom plate. The cells were rested overnight at 37°C, 5% CO₂, in 200 μL of RPMI 1640 supplemented with 10% FBS. The plate was then centrifuged and media replaced with 50 μL of fresh MAIT cell stimulation media, or stimulation media harvested as supernatant from MAIT cells stimulated as above (“MAIT S/N”). The HT‐29 cells were incubated for 60 min at 37°C, then fixed and permeabilized before staining for pSTAT6 (pSTAT6‐PE, clone pY641; BD Bioscience). STAT6 phosphorylation was determined by analysis of mean cell fluorescence on an LSR Fortessa (BD Biosciences). Similar assays were established that included purified anti‐IL‐13 (LEAF, clone JES10‐5A2; BioLegend) at a concentration of 10 μg mL⁻¹ (MAIT S/N + α‐IL‐13). Controls included HT‐29 cells cultured in RPMI + FBS (Media), 30 ng mL⁻¹ recombinant IL‐13 (carrier free; BioLegend) in RPMI + 10% FBS (“IL‐13”) or 50 μL of 30 ng mL⁻¹ recombinant IL‐13 in RPMI + FBS + 10 μg mL⁻¹ anti‐Il‐13 (IL‐13 + α‐IL‐13) (Figure 4).