Tumor-Infiltrating Leukocyte Profiling

Tumor-infiltrating leukocytes were isolated form breast tumors and investigated for different cell surface and intracellular markers with flow cytometry [12 (link)]. For that purpose, 1 × 10⁶ isolated tumor-infiltrating leukocytes were incubated with anti-mouse F4/80, CD11c, NK1.1, CD80, CD86, I-A, granzyme B, CD178, CD4, and CD8 monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (BD Biosciences, San Jose, CA, USA). For intracellular cytokine staining, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) and then incubated in a BD fixation/permeabilization solution (BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit) and washed in 1× BD Perm/Wash™ buffer. Fixed/permeabilized cells were stained for TNF-α, IFN-γ, IL-12, IL-4, IL-17, IL-10, and FoxP3 by using appropriate anti-mouse monoclonal antibodies conjugated with FITC, PE, PerCP, and APC (BD Biosciences, San Jose, CA, USA). BD Biosciences' FACSCalibur and Flowing Software were used for flow cytometry analysis [12 (link)].