Foxp3 fjk 16s staining

Prior to organ dissection, mice were perfused with 20 ml PBS to eliminate contaminating blood leukocytes. Single-cell suspensions of the pancreata were prepared by Collagenase P (Roche) digestion. Cells from pancreatic lymph nodes and spleen were prepared by physical dissociation. The spleen was treated with ACK lysing buffer (Thermo Fisher Scientific). All stainings began with an incubation with TruStain fcX anti-mouse CD16/32. Antibodies used for subsequent stainings were: anti-CD45 (30-F11), -CD19 (6D5); -CD3 (145–2C11), -CD4 (RM4–5), -CD8 (53–6.7), -CD25 (PC61), CD11b (M1/70), -CD11c (N418), -F4/80 (BM8), and -Gr1 (RB6–8C5) (all from BioLegend). Intracellular Foxp3 (FJK-16s) staining was performed according to eBioscience’s protocol. Samples were acquired with an Attune NxT flow cytometer (Thermo Fisher Scientific) and data were analyzed with FlowJo software (Tree Star, Inc.).

Single-cell suspensions were incubated with Fc receptor-blocking anti- CD16/CD32 antibodies (2.4G2) for 15 min at 4 °C. Cell suspensions were next stained with surface fluorescent-conjugated antibodies for 30 min at 4 °C. The following antibodies were used: CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD44 (1M7), CD25 (3C7), CD11b (M1/70), CD45 (30-F11), and MHC II (M5/114.15.2). For surface labeling of GGT1, unconjugated mouse monoclonal anti-GGT1 (1:100; ab55138, Abcam) was incubated with surface markers for 1 h at 4 °C. Then FITC-conjugated species-specific secondary antibody (1:100) was added for 1 h at 4 °C. For intracellular staining of IFN-γ and IL-17A, T cells were stimulated for 4 h with Cell Activation Cocktail (BioLegend). Cells were then fixed with Cytofix/Cytoperm solution (BD Bioscience) and stained with antibodies to IL-17A (TC11-18H10.1), or IFN-γ (XMG1.2). Foxp3 (FJK-16S) staining was performed according to the manufacturer’s protocol (eBioscience). Cells were analyzed by flow cytometry on an LSRII (BD Biosciences) with FlowJo software (Tree Star Inc.). Antibodies were purchased from BioLegend, BD Biosciences, or eBioscience and were used at 1:300 dilution unless stated otherwise.