Anti mouse foxp3 staining set apc

The BMDCs were incubated with mouse Fc block (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°. Fluorochrome‐labelled monoclonal antibodies used in the experiments included anti‐mouse CD80‐allophycocyanin (APC), CD86‐phycoerythrin (PE), MHC‐II‐PE, CD11c‐APC, CD40‐APC, CD83‐PE, CCR7‐PE, CCR5‐APC, CXCR3‐APC, programmed death‐ligand 1 (PD‐L1) ‐PE, CD3e‐FITC, CD4‐PE, CD8‐APC, IL‐17‐PE, IL‐10‐PE, interferon‐γ (IFN‐γ) ‐PE‐Cy7, Foxp3‐APC and CD25‐PE (eBioscience) and their corresponding isotypic controls. For intracellular cytokine staining, cells were pre‐treated with Golgi Stop (BD Biosciences, Franklin Lakes, NJ) and were then stained with fluorochrome‐labelled surface staining monoclonal antibodies or isotypic control for 30 min. A Cytofix Cytoperm kit (BD Biosciences) was then used to permeabilize and fix cells at 4° for another 30 min. Perm wash buffer was used to wash the processed cells before staining them with anti‐IFN‐γ, anti‐IL‐17 and anti‐IL‐10 monoclonal antibodies and isotypic controls for 30 min at 4°. Anti‐mouse Foxp3 staining set APC (eBioscience) was used for Foxp3 immunostaining. Data were captured by flow cytometry (Beckman Coulter, Brea, CA) and analysed using the flowjo software (FlowJo, Ashland, OR).

Immune cells were isolated from the kidneys digested with collagenase IV (Sigma, USA) followed by Ficoll-Paque density gradient centrifugation. Splenocytes were isolated as described earlier [27] (link) from which CD11b+ monocytes were extracted by magnetic isolation (Miltenyi Biotec, USA). Cells were incubated with mouse Fc block (Miltenyi) at 4°C followed by fluorochrome-labelled monoclonal antibodies and corresponding isotypic controls: anti-mouse CD3-APC, CD4-PE, CD4-APC, CD4-FITC, CD8-APCCy7, CD11c-FITC, CD11b-PE, CD25-PE, ST2-APC, CD206-PECy7 (BioLegend), CD19-PECy7, inducible nitric oxide synthase (iNOS)-APC (eBioscience, USA). For intracellular staining for interferon (IFN)-γ and IL-17, splenocytes were stimulated ex vivo by phorbol myristate acetate (50 ng/mL, Sigma) and ionomycin (1 μg/mL, Sigma) for 4 h in the presence of GolgiStop (eBioscience). After surface staining, Cytofix/Cytoperm kit (BD Biosciences) was used to permeabilise cells before staining with anti-IFN-γ-FITC, anti-IL-17A-PECy7, anti-iNOS-APC monoclonal antibody, and isotypic controls. Anti-mouse Foxp3 staining set APC (eBioscience) was used for Foxp3 immunostaining. Data were captured by flow cytometry (Beckman, USA) and analysed by the FlowJo software (Tree Star, USA).