Pe labeled anti cd8a

Mouse BMDCs, or THP-1 cells, were stained with the following surface markers; FITC-CD11c, PE-CD40, PE-CD80, PE-86, PE-MHC-I, PE-CD83, or PE-CCR7 (BioLegend). Human DCs were stained with FITC-labeled anti-HLA-DR, PE-labeled anti-CD80, and FITC-labeled anti-CD86 (Miltenyi). Cell activation was analyzed using a FACSCalibur with CELLQuest (Becton Dickinson, New Jersey, USA). To assess CD8⁺ T cell generation in vivo, splenocytes from treated or vaccinated mice were stained with PE-labeled anti-CD8a (BioLegend) and FITC-labeled anti-IFN-γ (BioLegend). FITC-labeled IFN-γ staining was performed using an intracellular staining kit (BD Bioscience).

To detect the CD3⁺CD8⁺ T lymphocytes produced by the spleen and infiltrated in the tumor tissues, T lymphocytes were harvested from immunized mice. After the lysis of erythrocyte and passage through a 70 μm filter, the purified splenic T cells (5 × 10⁶ cells·mL⁻¹) were labeled with FITC-labeled anti-CD3e (Clone 145-2C11, Biolegend, San Diego, CA, USA) and PE-labeled anti-CD8a (Clone 53-6.7; Biolegend, San Diego, CA, USA) for 60 min at room temperature. After washing twice with PBS and resuspending in 1 mL PBS with 10% FBS, the stained cells were analyzed by flow cytometry (FCM). The number of CD3⁺CD8⁺ T cells was then quantified using a FACSCalibur with CellQuest software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA).