Fcs express 4 cytometry software

Cells were collected from the peritoneal cavity of treated mice by lavage with 5 ml sterile PBS/BSA1%/heat inactivated FCS2%/0.05% sodium azide. Pancreatic lymph nodes (PLNs) from treated mice were harvested into RPMI (Life Technologies, Australia). Single cell suspensions from both were blocked with anti-CD16/32 mouse Fc Block (BD Pharmingen, Australia) and analysed for the expression of cell surface markers using combinations of the following antibodies: CD3 (SK7), CD4 (L3T4), CD8a (53-6.7), B220 (RA3-6B2), F4/80 (BM8), CD25 (7D4), PD-L1 (M1H5) or CD19 (1D3) (BD Pharmingen or Life Technologies, Australia). For the identification of regulatory T cells, expression of the intracellular marker, Foxp3, was quantified using a mouse Foxp3 intracellular staining kit (BD Pharmingen, Australia). Appropriate isotype control antibodies were used. Labelled cells were analysed using the BD LSRII flow cytometer (BD Biosciences). Data were analysed using FCS Express 4 Cytometry software (De Novo Software). Gating strategies are shown in Fig S1.

A Mouse IL-10 Secretion Assay (Miltenyi Biotec, Australia) was used to identify and quantify the IL-10 secreting cells within the peritoneal cavity and the pancreatic lymph nodes (PLNs) of mice treated with FhES or PBS. In preparation for the assay, single cell suspensions of PLNs harvested from treated mice were cultured overnight in RPMI with 10% v/v heat inactivated FCS (Life Technologies, Australia). Peritoneal cells were harvested by lavage and analysed immediately using the IL-10 secretion assay. Initially, cells were labelled with a capture antibody specific for mouse IL-10, then returned to culture for 45 min at 37°C in RPMI with 10% v/v heat inactivated FCS. Cells were then stained with an IL-10 detection antibody or isotype control antibody, before being counterstained for cell surface markers CD19 or F4/80 to identify B cells and macrophages, respectively. Dead cells were excluded using Dapi staining (Life Technologies, Australia). Labelled cells were analysed using the BD LSRII flow cytometer (BD Biosciences). Data were analysed using FCS Express 4 Cytometry software (De Novo Software).