Anti mouse cd3 pe

The expression of α-gal epitopes was evaluated on SKOV3-gal cells by flow cytometry. Approximately 1 × 10⁶ parental SKOV3 and α1,3GT transfected cells were suspended in 2 % BSA/PBS and incubated with biotinylated bandeiraea simplicifolia isolectin B4 (BS-IB4) lectins, which specifically binds to α-gal epitopes. Then cells were incubated with PE-Cy5 conjugated streptavidin.
The effect of cancer cell vaccine on the immune system of the KO mice was evaluated by analyzing the changes of CD3 + CD4+ and CD3 + CD8+ T cells in spleen. First the spleen was removed, then cut into pieces and grinded gently through a 200 mesh sterile nylon net. The cell suspension was carefully collected and layered on the Ficoll-Paque, PREMIUM (GE Healthcare Life Sciences, USA). The separated splenic mononuclear cells were incubated with fluorochrome-conjugated antibodies directed at the following CD markers: PE anti-mouse CD3, FITC anti-mouse CD4, and PE-Cy7 anti-mouse CD8 (all purchased from eBioscience, San Diego, CA). Gated CD3 positive events were analyzed for CD8+ and CD4+ T cells. Flow cytometry was performed using a FC500 flow cytometer (Beckman Coulter, Fullerton, CA) and analyzed using Beckman Coulter CXP software.

T lymphocytes were harvested from tumor-draining lymph nodes or spleen of tumor-bearing mice treated with different vaccines 48 h after last injection. T lymphocytes (1 × 10⁷) were stained with 200 nM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technology, USA) as per manufacturer’s instructions. CFSE-labeled T cells (3 × 10⁵) were incubated with 10 μM GPC3 epitopes (40 μg/ml) for 72 h as reported previously [29 (link)]. Subsequently, APC-cy7-anti-mouse CD45 (1:1000, BioLegend, USA), PE-anti-mouse CD3 (1:500, eBioscience, USA), Percp-cy5.5 or APC-anti-mouse-CD8α (1:500, BioLegend, USA) and PE-cy7-anti-mouse-IFNγ (1:500, BioLegend, USA) antibodies were used to counter-stain antigen-specific T cells at 4 °C for 30 min, followed by flow cytometric (BD LSRFortessa™ cell analyzer, USA) analyses.

For detecting macrophage/monocytes, DCs, neutrophils^{61 (link)} and CD8 T cells^{62 (link)}, 0.5 × 10⁶ cells were stained with anti-mouse CD8-FITC, anti-mouse CD49b-FITC, anti-mouse CD11b-phycoerythrim (PE), anti-mouse CD11c-APC-Cy7 (for DC exclusion) or CD11c-Per-CP (for activated DC population analysis) and anti-mouse Ly6G-APC-Cy7. To detect activated macrophage and DC populations, anti-mouse CD40-APC or OX40L-APC antibodies were also added (all from eBioscence). To detect activated T cells, anti-mouse CD3-PE, anti-mouse CD4-PerCP, anti-mouse CD8-FITC and anti-mouse OX40-APC or anti-mouse CD25-APC antibody (all from eBioscience) staining was performed for 30 min at 4 °C after blocking with 10% FCS in PBS with 10 mM EDTA, then fixed with 2% paraformaldehyde in PBS with 10 mM EDTA. Cells were analysed using FACS Canto (Becton Dickinson) acquiring 20,000 leukocytes (gated according to forward and side scatters with doublets excluded according to FCS-A/FCS-H dot plots). FACS results were subsequently analysed with FACS Diva (Becton Dickinson). Corresponding unstained cell samples for each antibody were acquired to establish gating areas for positively staining cells.