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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.

Figure 1. Subway plot of data from a 35–color panel run on a 5–laser Cytek Aurora. Gating hierarchy is indicated by colored subway lines to indicate branching of cell types. Histograms at left and right show comparative tertiary marker expression on subsets of T cells, B cells, NK and NK T cells, and antigen presenting cells including monocytes, macrophages, basophils, and plasmacytoid and dendritic cells.  (Open accordion below to see larger, detailed subway plot.)

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. 

Cell type Grouping Product
T cell Primary CD3 Monoclonal Antibody (UCHT1), NovaFluor Blue 660–120S, eBioscience
CD4 Monoclonal Antibody (SK3), NovaFluor Blue 555, eBioscience
CD8a Monoclonal Antibody (OKT8), NovaFluor Blue 585, eBioscience
BD OptiBuild™ BUV615 Mouse Anti–Human γδ TCR (BD Cat. No. 751308)
Secondary CD25 Monoclonal Antibody (CD25–4E3), PE–Cyanine7, eBioscience
CD28 Monoclonal Antibody (CD28.2), NovaFluor Yellow 660, eBioscience
CD45RA Monoclonal Antibody (MEM–56), PerCP
CD127 Monoclonal Antibody (eBioRDR5), Super Bright 702, eBioscience
Brilliant Violet 785™ Anti–Human CD197 (CCR7) (BL Cat. No. 353230)
Tertiary CD194 (CCR4) Monoclonal Antibody (D8SEE), PE, eBioscience
CD279 (PD–1) Monoclonal Antibody (EH12.2H7), APC
NK cell Primary CD16 Monoclonal Antibody (3G8), NovaFluor Yellow 730, eBioscience**
CD56 (NCAM) Monoclonal Antibody (TULY56), Brilliant Ultra Violet 737, eBioscience
Secondary CD335 (NKp46) Monoclonal Antibody (9E2), PerCP–eFluor 710, eBioscience
NK & T cell Tertiary CD183 (CXCR3) Monoclonal Antibody (CEW33D), PE–eFluor 610, eBioscience
B cell Primary CD19 Monoclonal Antibody (HIB19), NovaFluor Yellow 690, eBioscience
CD20 Monoclonal Antibody (2H7), Brilliant Ultra Violet 496, eBioscience
Secondary CD24 Monoclonal Antibody (eBioSN3 (SN3 A5–2H10)), Alexa Fluor 700, eBioscience
IgD Monoclonal Antibody (IA6–2), FITC, eBioscience*
IgM Monoclonal Antibody (SA–DA4), Brilliant Violet 480, eBioscience
B & T cell Secondary CD27 Monoclonal Antibody (O323), NovaFluor Yellow 590, eBioscience
Tertiary CD38 Monoclonal Antibody (HIT2), eFluor 450, eBioscience
BD OptiBuild™ BUV563 Mouse Anti–Human CD39, Brillant™ Ultra Violet 563 (BD Cat. No. 748473)
BD OptiBuild™ BUV395 Rat Anti–Human (CD185) CXCR5, Brillant™ Ultra Violet 395 (BD Cat. No. 740266)
CD196 (CCR6) Monoclonal Antibody (R6H1), Super Bright 436, eBioscience
Monocyte & Macrophage Primary CD14 Monoclonal Antibody (MEM–15), NovaFluor Blue 610–30S, eBioscience
CD16 Monoclonal Antibody (3G8), NovaFluor Yellow 730, eBioscience**
CD11b Monoclonal Antibody (ICRF44), eFluor 506, eBioscience
CD86 (B7–2) Monoclonal Antibody (IT2.2), Alexa Fluor 647, eBioscience
Dendritic cell Primary BD Horizon™ BUV661 Mouse Anti–Human CD11c, Brillant™ Ultra Violet 661 (BD Cat. No. 612967)
HLA–DR Monoclonal Antibody (LN3), Brilliant Ultra Violet 805, eBioscience
Secondary CD1c Monoclonal Antibody (L161), Super Bright 645, eBioscience
CD123 Monoclonal Antibody (6H6), APC–eFluor 780, eBioscience
CD141 Monoclonal Antibody (JAA17), FITC, eBioscience*
Tertiary CD85g (ILT7) Monoclonal Antibody (eBio17G10.2 (17G10.2)), PerCP–Cyanine5.5, eBioscience
Leukocytes Tertiary CD95 (APO–1/Fas) Monoclonal Antibody (DX2), Super Bright 600, eBioscience
Live Primary LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation
All Blocking buffer Brilliant Stain Buffer
Blocking buffer CellBlox Plus Blocking Buffer
Beads UltraComp eBeads Plus Compensation Beads
Staining Buffer eBioscience Flow Cytometry Staining Buffer
* Either IgD FITC or CD141 FITC should be used to focus on B cells or dendritic cell subsets but not both.
** CD16 is listed twice to show its value in identifying NK cells, as well as monocyte/macrophage populations.

Order human PBMC immunophenotyping panel 35–color products

Cy™ is a trademark or registered trademark of GE Healthcare.

Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.

Brilliant Ultra Violet™ and Brilliant Violet™ are trademarks or registered trademarks of Becton, Dickinson and Company or its affiliates, and is used under license.

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