Anti mouse cd11b apc

Bone marrow (BM) cells were flushed from mouse femurs and tibiae and prepared as single-cell suspension after lysing the red blood cells. Peripheral blood mononuclear cells (PBMC) in mouse blood samples were isolated with Ficoll-Hypaque (Axis-Shield). All cells labeled with antibodies were immediately analyzed on a FACS Calibur (BD Biosciences, Mountain View, CA, USA).
For the analysis of MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, and anti-mouse Gr1-PE.
For the analysis of PMN-MDSCs and M-MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Ly6G-FITC, and anti-mouse Ly6C-PE.
For the analysis of immature or undifferentiated markers CD11c, F4/80, CD80, and MHCII on MDSCs, the appropriate number of cells was pre-incubated with optimal concentration surface marker antibodies (eBioscience), anti-mouse CD11b-APC, anti-mouse Gr1-PE or anti-mouse Gr1-FITC, and anti-mouse CD11c-FITC, anti-mouse F4/80-FITC, anti-mouse CD80-PE, or anti-mouse MHCII-PE.

Immediately after sacrifice, bone marrow cells were flushed with FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA) and 1 × 10⁶ cells were incubated with fluorescent-conjugated antibodies. Antibodies for flow cytometric analyses included: anti-mouse F4/80 FITC (Abcam, Clone A3-1), F4/80 APC (Biolegend, Clone A3-1), anti-mouse CD11B APC (eBioscience, Clone M1/70), CD206 PE (AbD Serotec, Clone MR5D3), anti-mouse GR-1 PEcy3 (BDBiosciences, Clone RB6-8C5) and CD86 PE (BDBiosciences, Clone GL1). C-FMS+ cells were identified by GFP expressed in the MAFIA mice. Matched isotype controls were used and pre-gating was performed excluding dead cells and debris in SCC x FSC graph and aggregates in FSC-W x FSC-H and SSC-W x SSC-H.
For macrophage analysis in tumor tissue, tumors were mechanically dissociated, followed by digestion in complete RPMI-1640 media supplemented with type I collagenase (5 mg/mL; Sigma-Aldrich), 0.5% FBS and 1% Penicillin-streptomycin for 4 hours. Cells were washed in PBS multiple times to eliminate collagenase and passed through 75μm cell strainer for single cell suspension. Viable cells were counted and 1 × 10⁶ cells incubated in FACs staining buffer containing combinations of antibodies or isotype controls. After washing, cells were analyzed on BD FACSAria™ III.

Curcumin, piperine, actin antibody and protein G magnetic beads were purchased from Sigma-Aldrich, Inc (St. Louis, MO), Dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Molecular Probes (Eugene, OR). The following antibodies were purchased from eBiosciences (San Diego, CA): anti-mouse-Ly6G and Ly6C PE, anti-mouse-CD11b APC, anti-mouse F4/80 FITC, anti-mouse CCR7 APC, anti-mouse Ly6G FITC, anti-mouse CD4 FITC, anti-mouse CD8 PE, anti-mouse Ly6C PE, isotype control antibodies. Annexin V Apoptosis kit, Bax 6A7 antibody, anti-mouse-IFN-γ APC were obtained from BD Biosciences (San Jose, CA). Biotinylated anti-Gr1, anti-Ly6C, anti-Ly6G and streptavidin microbeads were purchased from Miltenyi Biotech (Auburn, CA). Anti-mouse Dectin-1 was purchased from AbD Serotec. Clusterin antibodies were purchased from Upstate (Millipore, Billerica, MA). Clusterin siRNA and Scrambled negative control were purchased from OriGene (Rockville, MD). MitoTracker Red CMXRos, Alexa 488 anti-mouse secondary antibody and Lipofectamine 2000 were purchased from Invitrogen (Grand Island, NY).