Anti cd8a ly 2 microbeads

Male BALB/c mice aged 4–5 weeks were inoculated with CT26 cells to obtain tumor‐bearing mice according to the protocol of animal model. Two weeks after CT26 cells inoculation, mice were sacrificed and spleens were collected to filter through a 70 µm filter to prepare a single cell suspension. Then, mononuclear cells were obtained using a lymphocyte separation medium. MDSCs were isolated using the antimouse Gr‐1‐Biotin antibody and antibiotin MicroBeads (Miltenyi Biotech, Germany) according to the protocol of the MDSC‐kit. The purity of isolated MDSCs was identified by staining with antimouse CD11b‐FITC (BioLegend, USA) and antimouse Gr‐1‐PE/Cy7 (BioLegend, USA) antibodies and further detected by flow cytometry. For CD8⁺ T cells purification, normal BALB/c mice aged 4–5 weeks were sacrificed and spleens were collected to filter through a 70 µm filter to prepare a single cell suspension. Similarly, mononuclear cells were obtained using lymphocyte separation medium and CD8⁺ T cells were directly isolated using the anti‐CD8a (Ly‐2) MicroBeads (Miltenyi Biotech, Germany) according to the protocol. The purity of isolated CD8⁺ T cells was identified by incubating with antibodies of antimouse CD3‐PerCP/Cyanine 5.5 (BioLegend, USA) and antimouse CD8‐APC (BioLegend, USA) and measured by flow cytometry analysis.

CD8⁺ cells were enriched from spleen cell preparations via positive selection using anti-CD8a (Ly-2) microbeads (Miltenyi Biotech) and then CD45.2⁺CD3⁺CD8⁺ T cells were sorted by flow cytometry. 5x10⁴ cells were seeded in wells of a 96-well plate with 100μL RPMI 1640 medium supplemented with 1mM sodium pyruvate, 10mM HEPES, 50μM mercaptoethanol, and 10% FCS with 40ng/mL rmIL-15 (Peprotech). Alternatively, wells were coated with anti-CD3 (5μg/mL, clone 145-2C11) and anti-CD28 antibodies (1μg/mL, clone 37.51) at 4°C overnight and then cells were added with 20ng/mL rmIL-2 (R&D systems) for 2 or 3d.