Alexa fluor conjugated foxp3 antibody

To evaluate the proportion of Tregs, 1 × 10⁶ splenocytes from vaccinated mice were harvested and stained with PE-conjugated CD4 and FITC-conjugated CD25 antibodies for 30 min at 4°C. Fc block was added before incubation with surface antibodies. Then, the cells were washed and permeabilized with FACSTM Permeabilizing Solution 2 (BD Bioscience) for 30 min at room temperature. After washing twice, the cells were stained with an Alexa Fluor-conjugated Foxp3 antibody (Miltenyi Biotec) for 30 min at 4°C. The samples were acquired on a FACS Calibur cell sorter (Becton Dickinson) and the data analyzed using WinMDI Ver. 2.9 software.

To evaluate the proportion of Tregs and macrophages, the splenocytes of vaccinated mice were isolated 7 days after the final DC vaccination (day 39), and 1 × 10⁶ splenocytes from vaccinated mice were harvested, washed, and stained with surface-staining antibodies for Tregs (CD4-PE and CD25-FITC) and macrophages (CD11b-FITC and F4/80-PE) for 30 min at 4°C. Fc block was added before incubation with surface antibodies. The cells were washed and permeabilized with FACSTM Permeabilizing Solution 2 (BD Bioscience) for 30 min at room temperature. After washing twice, the cells were stained with an intracellular staining antibody for Tregs (Alexa Fluor-conjugated Foxp3 antibody; Miltenyi Biotec, Bergisch Gladbach, Germany) and macrophages (CD206-APC) for 30 min at room temperature. The samples were acquired on a FACS Calibur cell sorter (Becton Dickinson) and the data were analyzed using WinMDI Ver. 2.9 software.

To evaluate the proportion of Tregs and macrophages, 1 × 10⁶ splenocytes from vaccinated mice were harvested, washed, and stained with surface-staining antibodies of Tregs (CD4-PE and CD25-FITC) and macrophages (CD11b-FITC and F4/80-PE) for 30 min at 4°C. Fc block was added before incubation with surface-staining antibodies. Next, the cells were washed and permeabilized with Fixation/Permeabilization Solution 2 (eBioscience) for 30 min at room temperature. After washing twice, the cells were stained with an intracellular staining antibody, Tregs [Alexa Fluor-conjugated Foxp3 antibody (Miltenyi Biotec)] and macrophages (CD206-APC) for 30 min at room temperature. The samples were acquired on a FACSCalibur cell sorter (Becton Dickinson), and data were analyzed using WinMDI ver. 2.9.