Intracellular Cytokine Profiling of Immune Cells

Intracellular cytokine expression was measured as described previously.⁷ (link) Cells were stimulated with IL23 (10–20 ng/mL), IL6 (10–100 ng/mL), or phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) and ionomycin (1 μmol/L) for 4–6 hours at 37°C with monensin (3 μmol/L) added for the last 2 hours. In human work, antibodies used to stain cell surface antigens were incubated with unstimulated cells for 25 minutes and then fixed in 2% paraformaldehyde pending analysis. For fluorescence-activated cell sorter purification of murine ILCs, CD45⁺ cells first were sorted immunomagnetically from unfractionated cLPMCs using anti-CD45 beads (Miltenyi) and LS columns (Miltenyi). CD45⁺ cells were stained with CD90, NKp46, and IL7R. Antibodies used in flow cytometry experiments are listed in Supplementary Table 1.