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. 2019 Feb 26;10:183. doi: 10.3389/fimmu.2019.00183

Figure 2.

Figure 2

Characterization of cultured MDSCs. (A) FACS profiles of cells cultured with human GM-CSF, G-CSF, or M-CSF (100 ng/ml) and human SCF (50 ng/ml) for 6 weeks. Blue histogram lines represent the GM-CSCF/SCF (MDSCs), light green histogram lines the cells cultured G-CSF/SCF, orange histogram lines the cells cultured M-CSF/SCF and pink histogram lines represent the adult mature DCs. (B) The cells were intracellular stained with anti-human MPO antibody and analyzed by flow cytometry. (C) The cells were lysed and fixed in a single step using Phosflow Lyse/Fix buffer for 10 min at 37°C. Cells were then permeabilized in Phosflow Perm Buffer for 30 min on ice. Cells were then washed twice in the stain Buffer and stained with fluorochrome conjugated anti-Stat (AF647), anti-Stat3 (PE), anti-Stat6 (PerCP-Cy5.5), anti-Akt (PE) and anti-mTOR (PerCP-efluor718) antibodies for 30 min on ice. Cells were washed twice in the stain buffer. All samples were acquired on a BD LSR Fortessa and then analyzed using FlowJo 9.2.1. Data are mean ± S.E.M. of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.