*2.12. IBD Patient PBMC Experiments*

This study received ethical approval from St Vincent's University Hospital Ethics and Medical Research Committee to take blood samples from consenting patients (*N* = 14) attending a specialist outpatient clinic for inflammatory bowel disease (IBD). PBMC were isolated as above and frozen at −80 ◦C. PBMC were thawed and treated with either IP or HPP (250–1000 μM) for 6 h prior to stimulation with anti-CD3 (1 μg/mL) for 12 h. The media was then removed and replaced with fresh media to circumvent issues that occur as the compounds become fluorescent after incubations over long periods of time, and cells were maintained in the presence of anti-CD3 for a further four days. The supernatants were then removed for analysis of IL-10, IFNγ, and IL-17A by ELISA. PBMC were restimulated in complete IMDM medium in the presence of 50 ng/mL PMA, 500 ng/mL ionomycin, and 5 μg/mL brefeldin A for 4 h. The cells were washed in PBS and stained for viability (Zombie NIRTM Fixable Viability kit) for 15 min at room temperature. The cells were then washed in PBS and stained with fluorochrome-conjugated antibodies for surface markers CD3 and CD8 for 15 min at room temperature. After this, the cells were then washed and fixed (FIX & PERM™ Cell Permeabilization Kit) for 15 min at room temperature. Finally, the cells were washed and stained for intracellular markers Ki67, IFNγ and IL-17 in permeabilization buffer for 15 min at room temperature. The cells were then washed and acquired on a BD LSRFortessa flow cytometer. The analysis was performed with FlowJo v.10. All antibodies used in this experiment were chosen carefully to avoid channels which still had some fluorescence issues, despite the steps taken to overcome this.
