Cd45 evolve655

Homeostatic stop^flox and stop^ΔIEC mice were sacrificed and colon tissues were harvested. Colon was flushed with cold PBS and fileted. Colon tissues were washed in cold PBS 3 times and in dPBS twice (vortexing for 1min each time). Tissues were digested in HBSS containing 1mM EDTA and 1mM DTT for 30min at 30°C with shaking. Tissues were vortexed for 1min and filtered through a 40 μm filter to obtain epithelial cell fraction. Epithelial cells were washed in HBSS (3 times) and resuspended in dPBS. Cells were stained withCD326 APC (EpCAM; ThermoFisher), CD45 evolve655 (ThermoFisher) and Zinpry-1 FITC (Cayman Chemical company) and analyzed by flow cytometry.

Cells were stimulated for 4 hours at 37°C in RPMI complete containing 1X cell stimulation cocktail (eBioscience) and protein transport inhibitor cocktail (eBioscience). Stimulated cells were harvested and stained for surface markers and viability for 30 min at room temperature. Samples were stained with viability dye APCef780 (ThermoFisher), CD45 evolve655 (ThermoFisher), CD4 BV785 (BioLegend), CD3 eF450 (ThermoFisher), CD8a BV605 (BioLegend), CD335 BV510 (BioLegend) and Gr-1 FITC (eBioscience). Following overnight fixation using the Fixation/Permeabilization solution (eBioscience Foxp3 Staining Buffer Set), cells were permeabilized and stained for cytokines IL-17A AF488 (eBioscience), IL-22 PE (eBioscience) and IFNγ PE (eBioscience). Cells were analyzed using the Aria IIIu cytometer and data was analyzed using the FlowJo software.