Figure 3.

MDSCs express immune suppressive molecules. (A) The cells cultured with each cytokine combinations for 6 weeks or (B) The cells cultured with each cytokine combinations from 4 to 6 weeks were fixed in Cytofix buffer for 10 min at 37°C and then permeabilized in Cytoperm buffer for 30 min on ice. Cells were then washed twice in the stain buffer and stained with FITC anti-iNOS antibody, PE anti-IDO antibody, and PerCP-Cy5.5 anti-arginase 1 antibody 30 min on ice. Cells were washed twice in the stain buffer and acquired on a BD LSR Fortessa. (C) The cells cultured with GM-CSF/SCF were stimulated with or without anti-CD40 antibody for 48 h and human VEGF, TGF-β and IL-10 were compared in culture supernatants between 3 and 6weeks. Data are mean ± S.E.M. of three independent experiments, each performed in triplicate. ***p < 0.001, ****p < 0.0001.