Whole-skin punch biopsies and cervix samples were incubated in 5U dispase (Life Technologies) overnight at 4°C followed by manual separation of epidermis and cervical epithelium from dermis or cervical submuocsa respectively followed by 90 min incubation in collagenase III (3 mg/ml; Worthington) with DNase (5 μg/ml; Roche) in RPMI 1640. Epidermal cell suspension was prepared by repeated pipetting. Dermis and submucosa were further processed by Medicon tissue disruptor (BD Biosciences) as previously described (Cheuk et al., 2014 (link)). Ileum biopsies were digested in collagenase II (0.25 mg/ml; Sigma-Aldrich) with DNase (0.2 mg/ml; Roche) in IMDM (Life Technologies) for 30–45 min. Complete RPMI medium was added and the cell suspension were subsequently passed through a 40 μm (gut) / 70 μm (skin or cervix) cell strainer (BD Bioscience). Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll (GE Healthcare) density separation.
P815 cells were purchased from ATCC and maintained in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum [FBS], L-glutamine; all Hyclone). Recombinant IL-15, IL-1β, IL-2, IL-6, IL-7, IL-23, IL-12 and IFN-α (all R&D Systems) were stored at −80°C. Human collagen IV, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma.
P815 cells were purchased from ATCC and maintained in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum [FBS], L-glutamine; all Hyclone). Recombinant IL-15, IL-1β, IL-2, IL-6, IL-7, IL-23, IL-12 and IFN-α (all R&D Systems) were stored at −80°C. Human collagen IV, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma.