Foxp3 pe nrrf 30

Cell suspensions were stained with combinations of following monoclonal fluorescently conjugated antibodies: TCRβ eFluor^® 450 (H57-597, eBioscience, San Diego, CA, USA), CD4 FITC (GK1.5, eBioscience), CD8a PerCP-Cyanine5.5 (53–6.7, eBioscience), CD25 APC (PC61.5, eBioscience), FoxP3 PE (NRRF-30, eBioscience), CD45R/B220 APC-Cyanine7 (RA3-6B2, Biolegend, San Diego, CA, USA), IgD Brilliant violet 650™ (11-26c.2a, Biolegend), CD11b Alexa Fluor^® 700 (M1/70, Biolegend), F4/80 PE (BM8, Biolegend), PD-1 PE-eFluor™ 610 (J43, eBioscience), CTLA-4 PerCP-Cyanine5.5 (UC10-4B9, Biolegend), IL-10 PerCP-Cyanine5.5 (JES5-16E3, Biolegend), LAP PerCP-Cyanine5.5 (TWT-16B4, Biolegend), CD14 FITC (Sa2-8, eBioscience), CD206 APC (MR6F3, eBioscience), MHC-II e Fluor^® 450 (M5/114.15.2, eBioscience). To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with intracellular fixation and permeabilization buffer kit (eBioscience) according to the manufacturer’s recommendation. For intracellular IL-10 staining, cells were incubated with PMA (50 ng/ml) and ionomycin (1000 ng/ml) for 2 h, then incubated with Brefeldin A (10.6 μM) and Monensin (2 μM) for 3 h. Data were acquired using BD Fortessa X20 (BD Bioscences, San Diego, CA, USA) and analyzed with FlowJo 7.6 software.

Lymphocytes were isolated from the C57BL/6 mouse spleens and MLNs in single-cell suspensions. The cells were stained with fluorescence-labeled antibodies against CD4-FITC and CD44-APC at 4°C for 30 min. The samples were stained with antibodies against Foxp3-PE (NRRF30, eBioscience Inc., San Diego, CA, USA) at room temperature for 30 min in the dark after using a Foxp3 fixation/permeabilization kit (BD Biosciences, San Diego, CA, USA) [29 (link)]. The lymphocytes collected from the Foxp3^GFP mouse spleens were stained with fluorescence-labeled antibodies against CD4-PE and CD62L-APC at 4°C for 30 min. Flow cytometry was performed on an LSRII cytometer (BD Biosciences) and analyzed with the FlowJo 7.6 software. Anti-mouse CD4-FITC (GK1.5) and Foxp3-PE (NRRF30) were purchased from eBioscience. CD62L-APC (MEL-14) was purchased from BD Biosciences. CD4-PE (GK1.5) and CD44-APC (IM7) were obtained from BioLegend Inc. (San Diego, CA, USA).