Human il 22

HT-29 cells (a gift from Lidia Sambucetti) were cultured in RPMI with 10% FBS, 25 mM HEPES (Life Technologies), and 2 mM Glutamax. Cells were serum-starved overnight (RPMI). Cells were plated at 2.5×10⁵ cells per well in a 96-well plate in starvation medium and incubated with SAR-20347 for 20 minutes (final concentration 0.5% DMSO) at 37°C, 5% CO₂. Cells were stimulated with 10 ng/mL human IL-22 (Peprotech) for 15 minutes, rinsed, and fixed in 4% paraformaldehyde for 10 minutes at room temperature. Cells were rinsed with 0.5% BSA in PBS, resuspended in 90% methanol, and incubated for 30 minutes on ice. Cells were stained with rabbit anti-pSTAT3 (1:200; Cell Signaling) for 1 hour at room temperature, rinsed in 0.5% BSA/PBS, then incubated in goat anti-rabbit Alexa488 (1:200; Invitrogen) for 30 minutes at room temperature, then analyzed by flow cytometry and gated on FSC/SSC. The IC₅₀ was determined by calculating mean fluorescence intensity, then subtracting background (secondary antibody alone), and plotted relative to DMSO/IL-22 control (as 100%).