Murine Lymphocyte Phenotyping by Flow Cytometry

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).