Af488 conjugated anti cd11c n418

CD11c⁺ dendritic cells and TCRγδ⁺CXCR5⁺, TCRγδ⁺CXCR5⁻ cells were first enriched using CD11c microbeads or TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. For TCRγδ sorting, AF488-conjugated anti-CD11c (N418; 1:100; Biolegend) was used to exclude any remaining CD11c⁺ cells in order to prevent dendritic cell contamination. For uptake assay, TCRγδ⁺CXCR5⁻, TCRγδ⁺CXCR5⁺ and CD11c⁺ dendritic cells were incubated for 3 h at 37 °C with 50 μg ml⁻¹ of ovalbumin (OVA) coupled to Alexa Fluor 488 (Invitrogen) in a 96-well round-bottom plate. After incubation, cells were collected, thoroughly washed and analyzed by flow cytometry. For in vitro presentation assay, cells were first incubated overnight at 37 °C with 50 μg ml⁻¹ of either OVA_323–339 peptide (Invivogen), OVA protein (Sigma) or medium only (unloaded cells as control) in a 96-well round-bottom plate. On the next day, cells were thoroughly washed and incubated at 1:2 ratio (antigen-presenting cells:responder cells) with sorted naïve (CD4⁺CD62L⁺CD44⁻Foxp3⁻) cells from OT-II-Foxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. Proliferation was then analyzed by flow cytometry.

CD11c⁺ dendritic cells, TCRγδ⁺CXCR5⁺ and TCRγδ⁺CXCR5⁻ cells from WT mice were first enriched using CD11c microbeads or TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. For TCRγδ sorting, AF488-conjugated anti-CD11c (N418; 1:100; Biolegend) was used to exclude any remaining CD11c⁺ cells in order to prevent dendritic cell contamination. Cells were then incubated overnight at 37 °C with 50 μg ml⁻¹ of either OVA_323–339 peptide, OVA protein or medium only (unloaded cells as control) or 50 μg ml⁻¹ of OVA_323–339 peptide plus either 1 μM of the porcupine (PORCN) inhibitor (Wnt-C59, Tocris) or 20 μg ml⁻¹ of anti-Wnt8b monoclonal antibody (LSBio) in a 96-well round-bottom plate. On the next day, cells were thoroughly washed and incubated at 1:2 ratio (antigen-presenting cells:responder cells) with sorted naïve (CD4⁺CD62L⁺CD44⁻Foxp3⁻) cells from OT-II-Foxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. CXCR5 expression on CD4 T cells was then analyzed by flow cytometry.

TCRγδ⁺CXCR5⁺ and TCRγδ⁺CXCR5⁻ cells from WT mice were first enriched using TCRγδ isolation kit (all from Miltenyi Biotec) and sorted. AF488-conjugated anti-CD11c (N418; 1:100; Biolegend) was used to exclude any remaining CD11c⁺ cells in order to prevent dendritic cell contamination. Cells were then stimulated with plate-bound anti-CD3 (145–2C11; Biolegend) and anti-CD28 (PV-1; Bioxcell), both at 2 μg ml⁻¹, for 3 days at 37 °C in a 96-well round-bottom plate. Wnt-C59 at 1 μM was added to some wells to prevent Wnt ligand release. At the 3rd day of culture, supernatants were collected and 100 μl added to naïve (CD4⁺CD62L⁺CD44⁻Foxp3⁻) cells in a 96-well round-bottom plate and stimulated with plate-bound anti-CD3 and anti-CD28 (both at 2 μg ml⁻¹) at 37 °C. Three days later, CXCR5 expression on CD4 T cells was analyzed by flow cytometry.