Identification of human myeloid-derived suppressor cell (MDSC) subpopulations

Figure 1:  Identification of MDSC subsets. Example of staining with the MDSC Detection Kit (without optional APC-conjugated antibodies). PBMCs were prepared from fresh blood samples of healthy donors. Gradient centrifugation was performed less than 30 minutes after blood collection. Please note that working with fresh material is crucial. Delays between blood collection and analysis or poor handling of the blood sample might lead to increased numbers of low density PMN cells in samples. 
Cells were stained with the MDSC Detection Kit, human, following the recommended protocol. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. All results shown in this example were obtained from a selected healthy blood donor who had an elevated number of MDSCs.
Samples were gated on CD45 expression (region R1), single events (region R2), non-debris (region R3), and on viable cells (region R4) (A). Cells from region R4 were further separated into three subsets: SSC^high cells, which correspond to low density PMN-MDSCs (region R5), CD14⁺ cells (region R6), and SSC^low CD14^– cells (region R7). Low density PMN-MDSCs shown in region R5 were further separated into four subsets based on the expression of CD16 and CD11b (gates R8 to R11) (B). Cells from R6 were gated on CD14 and HLA-DR expression for identification of CD14⁺ HLA-DR^– monocytic MDSCs (M-MDSCs) (C). Cells from region R7 were gated on HLA-DR and CD33 expression for identification of HLA-DR^– CD33^int early MDSCs (e-MDSCs) (D).

Figure 2: Analysis of LOX-1 expression on PMN-MDSCs subsets. Example of staining with the MDSC Detection Kit and analysis of LOX-1 expression on PMN-MDSCs subsets. PBMCs were prepared as described and cells were stained with the MDSC Detection Kit, human, following the recommended protocol, with the addition of LOX-1-APC to the Staining Cocktail and REA Control-APC to the Control Cocktail. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. All results shown in this example were obtained from a selected healthy blood donor who had an elevated number of MDSCs.
Samples were initially gated as shown in figure 1A and B. CD11b⁺ CD16^– low density PMN-MDSCs from R8 were displayed and LOX-1⁺ cells were gated (A). B) CD11b⁺ CD16⁺ low density PMN-MDSCs from R9 were displayed and LOX-1⁺ cells were gated (B). CD11b^– CD16^– low density PMN-MDSCs from R10 were displayed and LOX-1⁺ cells were selected (C). 

Figure 3: Analysis of CD10 expression on PMN-MDSC subsets. Example of staining with the MDSC Detection Kit and analysis of CD10 expression on PMN-MDSC subsets. PBMCs were prepared as described and cells were stained with the MDSC Detection Kit, human, following the recommended protocol, with the addition of CD10-APC to the Staining Cocktail and REA Control-APC to the Control Cocktail. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. All results shown in this example were obtained from a selected healthy blood donor who had an elevated number of MDSCs.
Samples were initially gated as shown in figure 1A and B. CD11b⁺ CD16^– low density PMN cells from R8 were displayed and CD10⁺ cells were gated (A). CD11b⁺/CD16⁺ low density PMN-MDSCs from R9 were displayed and CD10⁺ cells were gated (B). CD11b^– CD16^– low density PMN-MDSCs from R10 were displayed and CD10⁺ cells were selected (C).

Figure 4: Pre-gating of PMN-MDSCs with CD66b-APC. Example of staining with the MDSC Detection Kit and pre-gating of PMN-MDSCs with CD66b-APC. PBMCs were prepared as described and cells were stained with the MDSC Detection Kit, human, following the recommended protocol, with addition of CD66b-APC to both Staining and Control Cocktails. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. All results shown in this example were obtained from a selected healthy blood donor who had an elevated number of MDSCs.
Samples were initially gated as shown in figure 1A. Cells from region R4 were further separated into three subsets: CD66b⁺ cells, which correspond to low density PMN-MDSCs (region R12), CD14⁺ cells (region R13), and CD66b^– CD14^– cells (region R14). Low density PMN-MDSCs shown in region R12 were further separated into four subsets based on the expression of CD16 and CD11b (A). Cells from region R13 were gated on CD14 and HLA-DR expression for identification of CD14⁺ HLA-DR^– M-MDSCs (B). Cells from region R14 were gated on HLA-DR and CD33 expression for identification of HLA-DR^– CD33^int e-MDSCs (C).

Figure 5: Pre-gating of PMN-MDSCs with CD15-APC. Example of staining with the MDSC Detection Kit and pre-gating of PMN-MDSCs with CD15-APC. PBMCs were prepared as described and cells were stained with the MDSC Detection Kit, human, following the recommended protocol, with addition of CD15-APC to both Staining and Control Cocktails. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. All results shown in this example were obtained from a selected healthy blood donor who had an elevated number of MDSCs.
Samples were initially gated as shown in figures 1A. Cells from region R4 were separated into three subsets: CD15⁺ cells, which correspond to low density PMN-MDSCs (region R15), CD14⁺ cells (region R16), and CD66b– CD14^– cells (region R17). Low density PMN-MDSCs shown in region R15 were further separated into four subsets based on the expression of CD16 and CD11b (A). Cells from region R16 were gated on CD14 and HLA-DR expression for identification of CD14⁺ HLA-DR^– M-MDSCs (B). Cells from region R17 were gated on HLA-DR and CD33 expression for identification of HLA-DR^– CD33^int e-MDSCs (C).