Fix permeabilization kit

Flow cytometric analyses were performed using the following antibodies: anti-CD4, anti-CD11b, anti-CD33, anti-CD83, anti-CD86, anti-CD206 (BD Biosciences), anti-CD14, anti-CD58, anti-CD80, anti-HLA-DR (ImmunoTools), anti-CD16 (Thermo Scientific), anti-CD25, anti-GARP (eBioscience) and anti-Rab-32 (Abnova).
For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization kit (eBioscience) and stained with anti-Foxp3 mAb (BD Biosciences). Cytokine expression was analyzed in T cells re-stimulated with 50 ng/ml PMA plus 1 μg/ml Ionomycin for 5 h in the presence of Monensin (1.3 μM) 7 days after in vitro primary stimulation (day 0). Cells were then permeabilized as above and stained with anti-IL-2, anti-IFN-γ or anti-granzyme B mAb (BD Biosciences). Flow cytometry was performed on an LSRII FACS and FACSCalibur (BD Biosciences), using FlowJo software (Tree star).

After blocking FcR, cells were incubated with appropriately diluted antibodies and washed with PBS. For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization Kit (eBioscience, San Diego, CA, USA) and stained with antihuman Foxp3-APC mAb (BD Biosciences, San Diego, CA, USA, clone 259D/C7, cat num 560045). Acquisition was performed using FACSCanto II equipped with FACSDiva Version 6.1.3 (BD Biosciences). Data analysis was conducted using FlowJo Version 7.6.2 Software (Tree Star, Ashland, OR, USA). The antibodies used for surface staining in this study included antihuman CD4-PerCP/Cy5.5 (BD Bioscience, San Diego, CA, USA, clone RPA-T4, cat num 560650), antihuman GARP-PE (Miltenyi Biotec, Germany, cat num 130-103-889), and antihuman LAP (TGF-β)-APC (R&D, USA, cat num FAB2463A). The antibody used for intracellular staining included antihuman Foxp3-APC.

Flow cytometric analysis was performed using the following antibodies: Mouse IgG: anti-CD25 (M-A25, BD Biosciences), anti-CD4 (RPA-T4, BD Biosciences). For intracellular staining of Foxp3, cells were fixed and permeabilized using a Fix/Permeabilization kit (eBioscience) and stained with anti-Foxp3 mAb (anti-human Foxp3-PE, clone 259D/C7, BD Biosciences). EGFR was stained using biotinylated EGF according to the manufacturer’s instructions (R&D Systems). Flow cytometry was performed on LSRII and Accuri C6 (BD Biosciences), data were analyzed using FlowJo software (Tree star).