Fitc anti mouse cd11b

Splenocytes were cultured in RPMI 1640 medium (from Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and 50 µg/mL streptomycin (from Fisher Scientific, Waltham, MA). Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (Burlington, MA). The following antibodies were obtained from Biolegend (San Diego, CA) and used for flow cytometry analysis: FITC anti-mouse CD11b (clone: 29F.1A12), PE-CF594 anti-mouse F4/80 (clone:BM8), PerCPCy5.5 anti-mouse CD11c (clone: N418), PE anti-mouse CD86 (clone: GL-1), BV421 anti- mouse MHC-class II (clone: M5/114.15.2). The following antibodies were obtained from Biolegend (San Diego, CA) and used for flow cytometry analysis: FITC anti-mouse CD11b (clone: 29F.1A12), PE-CF594 anti-mouse F4/80 (clone: T45-2342), BV605 anti-mouse Ly6C (clone: HK1.4), BV711 anti-mouse Ly6G (clone: 1A8) PerCPCy5.5 anti-mouse CD11c (clone: N418), PE anti-mouse CD86 (clone: GL-1), BV421 anti- mouse MHC-class II (clone: M5/114.15.2).

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.

Single cells were prepared as previously described (Henry et al., 2009 (link); Xin et al., 2018 (link)) with minor modifications. Briefly, the ipsilateral cortex was quickly removed and immersed into PBS containing 0.2% BSA (w/v) on ice. The cortices of each group were gently ground on the 70 μm cell strainer at 4°C. Then, homogenates were filtered by passing through a 70 μm cell strainer. The cell suspension was centrifuged at 400 × g for 10 min to obtain precipitation, and the cells were re-suspended with 40% Percoll solution, followed by the addition of 75% Percoll solution at the bottom, and centrifuged at 500 × g for 20 min. Cells were collected from two density gradient interfaces and incubated by these antibodies for 30 min at 4°C: anti-mouse CD45-APC (10311, Biolegend) or anti-mouse CD11b-FITC (101206, Biolegend), to evaluate the populations of infiltrating monocytes/neutrophils (CD11b⁺/CD45^high cells). Flow cytometric analysis was performed with use of FACS flow cytometer C6 (BD Biosciences). CD11b⁺ cell populations were further gated for CD45 expression and divided into a low (microglia) and high population (infiltrating monocytes/neutrophils).