Human PBMC Immunophenotyping Panel 35–color

Complex panel design can be difficult and time consuming. Errors in the panel design can lead to wasted time and resources. Pre–optimized, experimentally verified backbone panels can shorten your workflow, and help reduce complexity and iteration when designing flow cytometry experiments.

The following subway plot (Figure 1) details a 35–color immunophenotyping flow cytometry panel. This panel shown has been designed and tested to allow for identifying and phenotyping a wide array of cells in the human peripheral blood immune system including gamma delta T, CD4 and CD8 conventional T cells, T regs, NK and NK T cells, B cells, monocytes and macrophages, plasmacytoid and conventional dendritic cells. It can be simplified into smaller panels to focus on your cells of interest, used as a backbone with primary and secondary markers, or to allow you to switch in your favorite markers of interest.

Figure 1 shows the subway plot of data from a 35–color panel run on a 5–laser Aurora showing hierarchical gating of human peripheral blood cell populations including the following cell populations (clockwise from the top): monocytes and macrophages; gamma delta, CD4 and CD8 conventional T cells; T regs; NK and NK T cells; B cells; basophils, plasmacytoid and conventional dendritic cells.

To obtain this data, 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 35–color 5–laser Cytek Aurora panel grouped by cell type expression for T and NK cells, B cells, and antigen presenting cells which include monocytes, macrophages and both conventional and plasmacytoid dendritic cells. 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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