Fitc anti gr 1

The following anti-mouse antibodies were from eBioscience: FITC-anti-F4/80, APC-anti-CD80, PE-Cy7-anti-CD86, PE-anti-MHC-II, PE-anti-CD11b, FITC-anti-Gr-1, APC-Cy7-anti-CD3, PerCP-Cyanine5.5-anti-CD4, APC-anti-CD8a, PE-anti-IFN-γ, and FITC-anti-IL-17. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml, Enzo Life Sciences) and ionomycin (1 nM, Enzo Life Sciences) in the presence of brefeldin A (1 mg/ml, Enzo Life Sciences) for 4–5 h. Surface staining was performed in FACS buffer (0.5% BSA, 2 mM EDTA, 0.02% sodium azide in PBS) in the presence of Fc receptor blocking antibody (BioLegend) for 20 min at 4°C. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature followed by permeabilization (0.1% BSA, 0.5% saponin in PBS) or with the BD Cytofix/Cytoperm Fixation and Permeabilization Kit. Cytokine staining was performed in permeabilization buffer for 20 min at 4°C. Data acquisition and analysis were performed using BD LSRFortessa X-20 (BD Bioscience) and FlowJo software (Tree Star, Ashland, OR, USA) respectively.