Isolation and analysis of macrophages and myeloid-derived suppressor cells (MDSCs)

Human PBMCs were stained using the MDSC Phenotyping Cocktail Kit, anti-human, REAfinity and analyzed by flow cytometry using the MACSQuant^®Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45⁺cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD^–cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter^high(SSC^high) cells, which correspond to low density PMN-MDSCs (region R5), CD14⁺cells (region R6) and SSC^lowCD14^–cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14⁺HLA-DR^–. D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^–CD33^int.

CD163⁺cells were isolated from human peripheral blood mononuclear cells (PBMCs) (A) or ovarian carcinoma samples (B) using the CD163 MicroBead Kit, an MS Column, and a MiniMACS™ Separator. Cells were fluorescently stained with CD45-VioBlue^®as well as with CD14-FITC and CD163-VioBlue (A) or CD206-FITC and CD163-PE (B) and analyzed by flow cytometry using the MACSQuant^®Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Isolation of F4/80⁺ macrophages.  F4/80⁺ cells were isolated from spleen single-cell suspension using Anti-F4/80 MicroBeads UltraPure, two MS Columns, and an OctoMACS™ Separator. The cells were fluorescently stained with CD45-VioBlue®, CD11b-APC, and Anti-F4/80-FITC and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and 7-AAD fluorescence.