Validated Flow Cytometry Panels
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Panels for 3-laser cytometers with violet (405 nm), blue (488 nm), and red (640 nm) lasers.
Planning flow cytometry experiments can be challenging, especially when new to the application. Careful consideration is needed for many aspects, including which markers to select, the antibody clone, appropriate controls, the protocol, and the gating strategy.
To help you start running simple 4-8 color panels, we have designed and optimized four human and one mouse immunophenotyping panels that enable you to detect major cell types in specific samples:
- Human Basic T and B Cell Panel
- Human Regulatory T Cell Panel
- Human T Cell, B Cell, NK Cell, NKT Cell, and Monocyte Panel
- Human Basic Myeloid Panel
- Mouse Basic T and B Cell Panel
These panels have been designed to work on three laser conventional cytometers configurations with violet (405 nm), blue (488 nm) and red (640 nm) lasers, and full spectrum instruments.
A staining protocol is provided for each panel with detailed antibody-fluorophore information. The use of viability dyes ensure dead cells are not included during the analysis. The gating strategy provided includes data from fluorescence minus one (FMO) controls, where appropriate to help accurately position gates on smaller populations. Example data is shown to demonstrate the quality of the data you can expect and also to provide a reference to check the appearance of your own panels.
These validated panels all have the same initial gating strategy:
- Step 1. Live cells gating — excludes dead cells that can give false positives due to high auto fluorescence and non-specific binding to antibodies
- Step 2. Single cell gating — excludes doublets that can cause misidentification of cells
- Step 3. CD45-positive cell gating — identifies hematopoietic blood cells enabling the same panel to be used for solid tissues as non-hematopoietic cells can be excluded
An example is shown in Figure 1.
Fig. 1. Gating steps to identify live, single hematopoietic cells in human peripheral blood. A, area; DAPI, 4’,6-diamidino-2-phenylindole; FSC, forward scatter; H, height; SSC, side scatter. FITC, SBV, StarBright Violet; W, width.
Human Basic T and B Cell Panel
A five-color antibody panel is described to enable identification of T and B cell populations in human peripheral blood. The attached pdf includes a detailed protocol on sample preparation, staining, compensation, and gating for this panel.
Top Tip: For easy panel expansion add in an additional antibody conjugated to StarBright™ Blue 580 or PE. For example, anti-CD56 to identify natural killer (NK) and NKT cells.
Antibodies used in the panel are shown in Table 1.
Target |
Fluorophore |
Laser |
Em max |
Catalog Number |
|---|---|---|---|---|
|
CD3 |
SBV440 |
405 |
436 |
|
|
CD4 |
SBV515 |
405 |
516 |
|
|
CD8 |
FITC |
488 |
525 |
|
|
CD45 |
SBB675 |
488 |
675 |
|
|
CD19 |
A647 |
640 |
665 |
Table 1. List of Bio-Rad antibodies used in the human basic T and B cell panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 or violet laser, those in blue by the 488 or blue laser, and those in red by the 640 or red laser. A647, Alexa Fluor 647; Em, emission; FITC, fluorescein isothiocyanate; SBB, StarBright Blue; SBV, StarBright Violet.
Example data, including the gating strategy used to identify T helper and cytotoxic T cells, and B cells (Figure 2). Percentages are shown but may differ between experiments due to variations between donors.
Fig. 2. Human basic T- and B-cell panel data. Red cell lysed human peripheral blood was stained with the basic human T- and B-cell panel and data acquired on a ZE5 Cell Analyzer. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). Cells were gated on live, single CD45+ cells prior to the gating strategy shown. A647, Alexa Fluor 647; FSC-A, forward scatter-area; FITC, fluorescein isothiocyanate; SBV, StarBright Violet; SSC-A, side scatter-area.
Human Regulatory T Cell Panel
A five-color antibody panel is described to enable identification of regulatory T cells in human peripheral blood. The attached pdf includes a detailed protocol on sample preparation, staining, compensation, and gating and fluorescence minus one controls for this panel.
Top Tip: A commonly used intracellular marker for identifying regulatory T cells is Foxp3. This protocol uses CD25 and CD127 expression on the surface of T cells, which enables a quicker and simpler surface staining protocol.
Antibodies used in the panel are shown in Table 2.
Target |
Fluorophore |
Laser |
Em Max |
Catalog Number |
|---|---|---|---|---|
|
CD45 |
SBV440 |
405 |
436 |
|
|
CD4 |
SBV515 |
405 |
516 |
|
|
CD127 |
FITC |
488 |
525 |
|
|
CD3 |
SBB580 |
488 |
582 |
|
|
CD25 |
SBR670 |
640 |
666 |
Table 2. List of Bio-Rad antibodies used in the human regulatory T cell panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 or violet laser, those in blue by the 488 or blue laser, and those in red by the 640 or red laser. Em, emission; FITC, fluorescein isothiocyanate; SBB, StarBright Blue; SBR, StarBright Red; SBV, StarBright Violet.
Example data, including the gating strategy used to identify CD127-/lowCD25+ regulatory T cells from the T helper cell (CD3+CD4+) subset (Figure 3). Percentages are shown but may differ between experiments due to variations between donors.
Fig 3. Human regulatory T cell panel data. Red cell lysed human peripheral blood was stained with the human regulatory T cell panel and data acquired on a ZE5 Cell Analyzer. Cells were gated on live, single CD45+ cells prior to the gating strategy shown. Data was were analyzed using FSC Express 7 (De Novo Software by Dotmatics). FITC, fluorescein isothiocyanate; FSC-A, forward scatter-area SBB, StarBright Blue; SBR, StarBright Red; SBV, StarBright Violet; SSC-A, side scatter-area.
Human T Cell, B Cell, NK Cell and NKT Cell, and Monocyte Panel
An eight-color antibody panel is described to enable identification of T cells, B cells, NK cells, NKT cells and monocytes in human peripheral blood. The attached pdf includes a detailed protocol on sample preparation, staining, compensation, and gating for this panel.
Top Tip: When using a cytometer with more than three lasers, add markers conjugated to fluorophores excited by the extra lasers. This helps keep compensation and spreading low.
Antibodies used in the panel are shown in Table 3.
Target |
Fluorophore |
Laser |
Em Max |
Catalog Number |
|---|---|---|---|---|
|
CD19 |
SBV440 |
405 |
436 |
|
|
CD45 |
SBV515 |
405 |
516 |
|
|
CD8 |
SBV790 |
405 |
782 |
|
|
CD56 |
A488 |
488 |
519 |
|
|
CD3 |
SBB580 |
488 |
525 |
|
|
CD14 |
SBB700 |
488 |
703 |
|
|
CD16 |
A647 |
640 |
665 |
|
|
CD4 |
A700 |
640 |
723 |
Table 3. List of Bio-Rad antibodies used in the human T cell, B cell, NK cell, NKT cell, and monocyte validated panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 or violet laser, those in blue by the 488 or blue laser, and those in red by the 640 or red laser. AXXX, Alexa Fluor; Em, emission; SBB, StarBright Blue; SBV, StarBright Violet.
Example data, including the gating strategy used to identify T cells, B cells, NK cells, and NKT cells from the lymphocyte gate and monocyte subtypes from the monocyte gate (Figure 4). Percentages are shown but may differ between experiments due to variations between donors.
Fig 4. Human T cell, B cell, NK cell, NKT cells, and monocyte panel data.
Red cell lysed human peripheral blood was stained with the human T cell, B cell, NK, NT cell, and monocyte pane,l and data acquired on a ZE5 Cell Analyzer. Cells were gated on live, single CD45+ cells prior to the gating strategy shown. Data were analyzed using FSC Express 7 (De Novo Software by Dotmatics). AXXX, Alexa Fluor; FSC-A, forward scatter-area; SBB, StarBright Blue; SBV, StarBright Violet; SSC-A, side scatter-area.
Human Basic Myeloid Panel
An eight-color antibody panel is described to enable identification of monocytes, neutrophils, eosinophils, basophils, T and B cells in human peripheral blood. The attached pdf includes a detailed protocol on sample preparation, staining, compensation and gating for this panel.
Top Tip: While the other panels can also be used to stain peripheral blood mononuclear cells (PBMCs), this panel requires whole blood to identify granulocyte populations, as PBMC processing removes granulocytes.
Antibodies used in the panel are shown in Table 4.
Target |
Fluorophore |
Laser |
Em Max |
Catalog Number |
|---|---|---|---|---|
|
CD3 |
SBV440 |
405 |
436 |
|
|
CD14 |
SBV515 |
405 |
516 |
|
|
CD19 |
SBV790 |
405 |
782 |
|
|
CD66b |
FITC |
488 |
525 |
MCA216F* |
|
CD123 |
PE |
488 |
578 |
|
|
CD45 |
SBB700 |
488 |
703 |
|
|
HLA DR |
A647 |
640 |
665 |
|
|
CD16 |
A700 |
640 |
723 |
Table 4. List of Bio-Rad antibodies used in the human basic myeloid validated panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 or violet laser, those in blue by the 488 or blue laser, and those in red by the 640 or red laser. AXXX, Alexa Fluor; Em, emission; FITC, fluorescein isothiocyanate; PE, Phycoerythrin; SBB, StarBright Blue; SBV, StarBright Violet. * Not currently available for purchase
Example data, including the gating strategy used to identify neutrophils, eosinophils and basophils from the granulocyte gate, T cells and B cells from the lymphocyte gate and monocyte subtypes from the monocyte gate (Figure 5). Percentages are shown but may differ between experiments due to variations between donors.
Fig 5. Human myeloid panel data.
Red cell lysed human peripheral blood was stained with the human basic myeloid panel and data acquired on a ZE5 Cell Analyzer. Cells were gated on live, single CD45+ cells prior to the gating strategy shown. Data were analyzed using FSC Express 7 (De Novo Software by Dotmatics). AXXX, Alexa Fluor; Em, emission; FITC, fluorescein isothiocyanate; FSC-A, forward scatter-area; PE, phycoerythrin; SBB, StarBright Blue; SBV, StarBright Violet; SSC-A, side scatter-area.
Mouse Basic T and B Cell Panel
A five-color antibody panel is described to enable identification of T and B cells in mouse peripheral blood. The attached pdf includes a detailed protocol on sample preparation, staining, compensation and gating for this panel.
Top Tip: Mouse peripheral blood was used for staining, but this panel is also effective for staining cells isolated from mouse tissue, such as splenocytes. Our protocol for cell isolation from mouse tissues can be found here.
Antibodies used in the panel are shown in Table 5.
Target |
Fluorophore |
Laser |
Em Max |
Catalog Number |
|---|---|---|---|---|
|
CD45 |
SBV440 |
405 |
436 |
|
|
CD45R |
SBV515 |
405 |
516 |
|
|
CD3 |
A488 |
488 |
519 |
|
|
CD4 |
SBB700 |
488 |
703 |
|
|
CD8 |
SBR670 |
640 |
666 |
Table 5. List of Bio-Rad antibodies used in the human basic myeloid validated panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 or violet laser, those in blue by the 488 or blue laser, and those in red by the 640 or red laser. A488, Alexa Fluor 488; Em, emission; SBB, StarBright Blue; SBR; StarBright Red; SBV, StarBright Violet.
Example data, including the gating strategy used to identify, T helper and cytotoxic T cells and B cells (Figure 6). Percentages are shown but may differ between experiments due to variations between donors.
Fig 6. Mouse basic T and B cell panel data. Red cell lysed mouse peripheral blood was stained with the mouse basic T and B cell panel, and data acquired on a ZE5 Cell Analyzer. Cells were gated on live, single CD45+ cells prior to the gating strategy shown. Data were analyzed using FSC Express 7 (De Novo Software by Dotmatics). A488, Alexa Fluor 488; Em, emission; FSC-A, forward scatter-area; SBB, StarBright Blue; SBR; StarBright Red; SBV, StarBright Violet; SSC-A, side scatter-area.
Best Practices
Although we have validated these panels, we still recommend following flow cytometry best practice due to differences in standard laboratory practices, instrumentation and samples.
- Use a high-quality sample
- Block Fc receptors
- Titrate antibodies and viability dyes
- Include appropriate controls
- Treat conjugated antibodies with care
Panel Expansion
These panels are ideal for using as a backbone panel, by providing a starting point to build bigger panels. For example, the memory status of T cells using CD45RA and CD45RO expression could be included as could activation markers such as CD69, or even combining with proliferation dyes such as CytoTrack. When building multicolor panels from scratch or when expanding an existing panel, panel design best practices should always be followed. An example of how these principles can be applied is shown in our 27 color technical note.