Anti mouse rat foxp3 fitc

The following antibodies were used for flow cytometry analysis: CD45-AF700, CD25-PE, CD127-APC-eFluor780, CD3-SuperBright436, CD19-eFluor506, CD11b-PE/Cy5, Ly6C-AF488, Ly6G/Ly6C(Gr-1)-APC, Ly-6G-PE, Anti-Mouse/Rat Foxp3 FITC, and isotype control (Rat IgG2a K Isotype Control FITC) were procured from eBioscience (Waltham, MA USA); CD4-PE-Cy7 and CD8a-APC700 from BD Biosciences (San Jose, CA, USA).
Isolated cells from the spleens or tumors were incubated with anti-CD16/CD32 antibodies (BD Biosciences San Jose, CA, USA) to prevent non-specific antibody binding. Surface antigens were stained with the antibodies diluted in PBS containing 5% FCS and 2 mM EDTA and maintained at 4 °C. Intracellular stainings were performed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, Waltham, MA, USA).
Initially, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen). All samples were fresh samples, and all cells quantified were live cells (data not shown). We decided, subsequently, not to use LIVE/DEAD Fixable Aqua Dead Cell Stain to free one more channel for use.
Multiparametric assessment was conducted utilizing a Gallios flow cytometer (Beckman Coulter, Villepinte France), with subsequent analysis executed through the Kaluza® software. Flow cytometric evaluation of immune cells was restricted to live CD45+ cells following the exclusion of doublets.

Lymphocytes were separated from spleen tissue samples using lymphocyte separation medium (Solarbio Biotec, Beijing). The cells were counted, and 5 × 10⁵ to 1 × 10⁶ cells were harvested in cold PBS. The cells were then resuspended in 100 μl PBS and incubated with 0.5 μg anti-mouse CD3a PE, 0.25 μg anti-mouse CD4 FITC, 0.125 μg anti-mouse IFN gamma PE, 0.25 μg anti-mouse IL-4 FITC, 1 μg anti-mouse/rat Foxp3 FITC, 0.25 μg anti-mouse IL-10 FITC or isotype control antibodies (eBioscience, San Diego, CA). The cells were then washed with cold PBS, and 10⁴ cells per sample were analyzed by flow cytometry (CyFlow, PARTEC).