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Our catalog makes it is possible to zoom in on monocytes, macrophages, and dendritic cells to more deeply phenotype professional antigen presenting cells. We have made use of our pre–optimized, experimentally verified 35–color immunophenotyping panel and generated this 15–color backbone panel. With our antigen presenting cell backbone, you can use the most common markers to identify monocytes, macrophages and dendritic cells and then add your preferred deeper markers based on the hypothesis you are testing. Since antigen presenting cells are often found in tissue, we have included 14 colors from the 35–color panel as well as CD45, a pan leukocyte marker to exclude any contaminating epithelial, endothelial or other non–immune cell populations.
The utility of this panel to identify a large number of antigen presenting cell populations is demonstrated in Figure 1. Live CD45+ CD3- CD19- cells were gated for non–T, non–B leukocytes to allow for identification of antigen presenting cells. CD56 and CD14 gating allowed for the exclusion of NK cells. Using CD14 and the HLA Class II antigen, HLA–DR, antigen presenting cell populations could be further subdivided. CD14-, HLA-DR–, CD123+ cells were classified as putative basophils, and CD14– HLA–DR+ cells were split into plasmacytoid dendritic cells (pDCs) and classical dendritic cells (cDCs) based on CD123 vs CD11c expression, respectively. cDCs were then further divided into cDC1 and cDC2 subpopulations using CD141 vs CD1c expression. In addition, monocytes and macrophages were allocated into classical (CD14+ CD16–), intermediate (CD14+ CD16+) and non–classical (CD14– CD16+) monocyte populations using CD14 and CD16 expression.
Figure 1. Gating of antigen presenting cell panel with singlets (top left), live cells (top second from left) and scatter populations (top third from left). Leukocytes were separated from non–lymphoid cells using CD45 gating (top right) and T, B, and NK cell populations were excluded using CD3 and CD19 (middle left) and CD56 (middle row second from left). For monocytes and macrophages, CD14 and CD16 populations (middle right) were gated on non–B, T, and NK populations and divided into non–classical (CD14– CD16+), intermediate (CD14+ CD16+) and classical (CD14+ CD16–) monocytes and macrophage (middle right). To identify dendritic cell populations, a broad scatter gate was used and then B cells, T cells and CD56+ NK cells were excluded from analysis (middle left first and second plot). Conventional dendritic cells were recognized as CD14– HLA–DR+ (middle second from right) to select for antigen presenting cells that were not monocyte derived. Plasmacytoid and conventional dendritic cells were separated based on CD123 and CD11c expression (bottom middle). Conventional dendritic cells (cDC) could be further identified based on CD1c and CD141 expression (bottom right) into cDC1 and cDC2 cDC populations. Putative basophils were identified as non–B, T, or NK cells that also lack expression of CD14, HLA–DR and CD11c but express CD123 (bottom left) (Click image to enlarge).
In Figure 2, the comparative expression of specific markers associated with antigen presenting cells was demonstrated. In Figure 2A–B, basophils are shown to express little of the antigen presenting molecules, HLA–DR and CD1c and do not express CD11c or CD86 (B7–2). In contrast, cDCs express high levels of HLA–DR, CD1c and intermediate levels of CD86 (B7–2). Monocyte and macrophage populations display higher levels of CD11b and CD86 but more varied levels of HLA–DR and CD1c antigen presenting molecules.
Figure 2. Comparative expression of specific markers on various subpopulations of antigen presenting cells using the monocyte and macrophage panel focused on (A) HLA–DR, CD1c, CD11b, CD11c, and CD123 and (B) CD86 respectively (Click image to enlarge).
Human PBMCs were stained and acquired on a 5–Laser Cytek Aurora with standard configuration, using the Cytek assay settings. CellBlox Blocking Buffer and Brilliant Stain Buffer were added to the antibody cocktail to block non–specific cell binding to NovaFluor dyes and polymer dye–dye interactions, respectively. Cells were stained with antibodies (Table 1) for at least 30 minutes at 4°C in the dark and fixed with 2% paraformaldehyde. Antibodies were titrated to determine optimal staining concentrations for cells. Single colors were generated on both cells and beads with the most optimal control (cells by default, beads if needed) used for unmixing raw data files. Spectral unmixing was performed using Cytek SpectroFlo software version 3.1.0. Analysis was performed using BD FlowJo version 10.8.1.
Table 1. Markers from 15–color 5–laser Cytek Aurora panel grouped by cell type expression for monocytes and macrophages, dendritic cells as well as exclusionary markers such as those for T, NK and B cells. The panel is made up of 14 markers from an experimentally–verified 35–color broad human immune panel as well as 1 additional marker in CD45 (see * in the table below). Since monocyte, macrophage, and dendritic cells can share markers with other cell types, CD45 is included to distinguish leukocytes from tissue resident non–immune cells and CD3, CD56, and CD19 are used to exclude T, NK and B cell populations respectively from the gating strategy. Primary markers refer to lineage markers that define populations and are typically highly and clearly expressed. Secondary markers refer to higher density markers with more continuous expression that typically subphenotype populations. Tertiary markers are typically expressed at lower levels.
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