Isolation and Culture of CD11b+CD33+CD14+HLA-DR-/lo (MDSC) and CD11b+CD33+CD14+HLA-DR+/hi (Monocytes)

Peripheral blood mononuclear cells were stained for CD11b, CD33, CD14, HLA-DR; CD11b⁺CD33⁺CD14⁺HLA DR^-/lo MDSC and CD11b⁺CD33⁺CD14⁺HLA DR^+/hi monocytes were isolated using Beckman Coulter MoFlo flow cytometer; sorted cells were >90% positive ( Supplemental Figure 1 ). Isolated cells (50, 000 – 80, 000) were cultured in RPMI 1640 (Gibco) and 10% human serum (MP Biomedicals) at 37⁰C and 5% CO₂ in the presence or absence of, M tuberculosis Erdman (Erdman) or M tuberculosis H37Rv whole cellular lysate (WCL) (10 µg/ml) (BEI resources) for 24 hrs. Supernatants and cells in Trizol were stored at -80°C for cytokine measurement and RNA purification, respectively. For some experiments, cells were cultured in the absence or presence of recombinant IL-27 (rIL-27) (10 ng/ml; R&D Systems).