Isolation of mononuclear cells from gastrointestinal tissue
Endoscopically obtained pinch biopsies were collected from Cohort 1
patients into Roswell Park Memorial Institute (RPMI) media containing
penicillin and streptomycin and 10% foetal calf serum. Biopsies
underwent enzymatic and mechanical digestion by being transferred into
5mL of warmed RPMI with penicillin and streptomycin/10% foetal calf
serum with 1mg/mL Collagenase D (Sigma-Aldrich, St Louis, USA) and
100µg/mL DNAse I (ThermoFisher, Waltham, USA) and shaken at 37°C for 1h.
Biopsies were then dissociated by vigorous agitation using a gentleMACS
Dissociator (Miltenyi Biotech, Bergisch Gladbach, Germany) and strained
through a 70µm strainer. Freshly isolated GMNC were analysed.