Human No Compensation Panels

The four major cell types in peripheral blood, T cells, B cells, monocytes, and granulocytes, can be identified using this panel.  Whole blood is required for this panel, as the preparation of PBMCs removes granulocytes. The antibodies used in the panel are shown in Table 1, and example data are shown in Figure 2.

Cell types identified:

• T cells: CD3+ CD19– CD14– CD11b–
• B cells: CD3– CD19+ CD14– CD11b–
• Monocytes: CD3– CD19– CD14+ CD11b+
• Granulocytes: CD3– CD19– CD14 lo CD11b+

Table 1. List of Bio-Rad antibodies and viability dye used in the T, B, and myeloid panel. Antibodies are colored according to the laser line: those in black are excited by the 355 nm or UV laser, those in blue by the 488 nm or blue laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser. Em, emission; SBR, StarBright Red; SBY, StarBright Yellow; SBUV, StarBright UltraViolet. 

Fig. 2. T, B, and myeloid cell panel data. Red cell lysed human peripheral blood was stained with the T, B, and myeloid cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single cells prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). SBR, StarBright Red; SBY, StarBright Yellow; SBUV, StarBright UltraViolet.

This panel identifies the four major cell types found in human peripheral blood lymphocytes: T cells, B cells, NK cells, and NKT cells. The antibodies used in the panel are shown in Table 2, and example data are shown in Figure 3.

Cell types identified:

• T cells: CD45+ CD3+ CD19–
• B cells: CD45+ CD3– CD19+
• NK cells: CD45+ CD3– CD19– CD56+
• NKT cells: CD45+ CD3+ CD19– CD56+

Table 2. List of Bio-Rad antibodies and viability dye used in the T, B, NK, and NKT cell panel. Antibodies are colored according to the laser line: those in black are excited by the 355 nm or UV laser, those in violet are excited by the 405 nm or violet laser, those in blue by the 488 nm or blue laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser. Em, emission; SBB, StarBright Blue; SBR, StarBright Red; SBY, StarBright Yellow; SBV, StarBright Violet.

Fig. 3. T, B, NK, and NKT cell panel data. PBMCs were stained with the T, B, NK, and NKT cell panel, and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). SBB, StarBright Blue; SBR, StarBright Red; SBY, StarBright Yellow; SBV, StarBright Violet.

CD3-positive T cells are composed of two primary subtypes: T helper cells and cytotoxic T cells. These groups can be identified using CD4 and CD8 markers. The antibodies used in the panel are listed in Table 3, and representative data are presented in Figure 4.

Cell types identified:

• T helper cells: CD45+ CD3+ CD4+ CD8–
• Cytotoxic T cells: CD45+ CD3+ CD4– CD8+

Table 3. List of Bio-Rad antibodies and viability dye used in the helper and cytotoxic T cell panel. Antibodies are colored according to the laser line: those in black are excited by the 355 nm or UV laser, those in violet are excited by the 405 nm or violet laser, those in blue by the 488 nm or blue laser, those in green by the 561 nm or yellow/green laser,  and those in red by the 640 nm or red laser. Em, emission; SBR, StarBright Red; SBUV, StarBright UltraViolet; SBV, StarBright Violet; SBY, StarBright Yellow.

Fig. 4. Helper and cytotoxic T cell panel data. PBMCs were stained with the helper and cytotoxic T cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). SBR, StarBright Red; SBUV, StarBright UltraViolet; SBV, StarBright Violet; SBY, StarBright Yellow.

When a naïve T cell first encounters an antigen, it transitions from expressing CD45RA to CD45RO as it becomes activated and forms memory cells. These markers help determine the memory status of T helper (CD4+) or cytotoxic (CD8+) T cells. Here, we present data for T helper cells; however, using a CD8 antibody, or including both CD4 and CD8 antibodies in the panel, allows assessment of cytotoxic T cell memory status as well. The antibodies used are listed in Table 4, and representative data are shown in Figure 5.

Cell types identified:

• T helper naïve cells: CD3+ CD4+ CD45RA+
• T helper memory cells: CD3+ CD4+ CD45RO+

Table 4. List of Bio-Rad antibodies and viability dye used in the naïve and memory T cell panel. Antibodies are colored according to the laser line: those in black are excited by the 355 nm or UV laser, those in violet are excited by the 405 nm or violet laser, those in blue by the 488 nm or blue laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser. Em, emission; SBR, StarBright Red; SBUV, StarBright UltraViolet; SBV, StarBright Violet; SBY, StarBright Yellow.

Fig. 5. Naïve and memory T cell panel data. PBMCs were stained with the naïve and memory T cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). SBR, StarBright Red; SBUV, StarBright UltraViolet; SBV, StarBright Violet; SBY, StarBright Yellow.

This protocol uses CD25 and CD127 expression on the surface of T cells to identify regulatory T cells (T regs). A commonly used alternative is FOXP3, an intracellular marker, here a cell permeabilization protocol would be required. The antibodies used in the panel are shown in Table 5, and example data are shown in Figure 6.

Cell types identified:

• T helper cells: CD3+ CD4+
• Regulatory T cells: CD3+ CD4+ CD127 lo CD25 hi

Table 5. List of Bio-Rad antibodies and viability dye used in the regulatory T cell panel. Antibodies are colored according to the laser line: those in black are excited by the 355 nm or UV laser, those in violet are excited by the 405 nm or violet laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser. Em, emission; PE, phycoerythrin; SBR, StarBright Red; SBV, StarBright Violet.

Fig. 6. Regulatory T cell panel data. PBMCs were stained with the regulatory T cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). PE, phycoerythrin; SBR, StarBright Red; SBV, StarBright Violet.

Naïve and memory B cells are distinguished by their differential expression of CD27 and IgD. The antibodies used in the panel are shown in Table 6, and example data are shown in Figure 7.

Cell types identified:

• Naïve B cells: CD19+ CD20+ CD27– IgD+
• Memory B cells: CD19+ CD20+ CD27+ IgD–

Table 6. List of Bio-Rad antibodies and viability dye used in the naïve and memory B cell panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 nm or violet laser, those in blue by the 488 nm or blue laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser. Em, emission; FITC, fluorescein isothiocyanate; SBB, StarBright Blue; SBR, StarBright Red; SBV, StarBright Violet.

Fig. 7. Naïve and memory B cell panel data. PBMCs were stained with the naïve and memory cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). FITC, fluorescein isothiocyanate; SBB, StarBright Blue; SBR, StarBright Red; SBV, StarBright Violet.

NK and NKT cells were identified by their expression of CD56, with NKT cells also expressing T cell markers, CD3 and CD4 or CD8. NK cells and a subset of NKT cells also express CD335. The antibodies used in the panel are shown in Table 7, and example data are shown in Figure 8.

Cell types identified:

• T cells: CD3+ CD45+ CD56– CD335–
• NK cells: CD3– CD45+ CD56+ CD335+
• NKT cells: CD3+ CD45+ CD56+

Table 7. List of Bio-Rad antibodies and viability dye used in the NK and NKT panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 nm or violet laser, those in blue by the 488 nm or blue laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser. A647, Alexa Fluor 647; Em, emission; FITC, fluorescein isothiocyanate; SBR, StarBright Red; SBV, StarBright Violet.

Fig. 8. NK and NKT panel data. PBMCs were stained with the NK and NKT panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). A647, Alexa Fluor 647; FITC, fluorescein isothiocyanate; SBR, StarBright Red; SBV, StarBright Violet.

Three different cell types have been identified in this panel: monocytes, granulocytes, and eosinophils. Neutrophils and basophils could also be identified with the addition of CD193. The antibodies used in the panel are shown in Table 8, and example data are shown in Figure 9.

Cell types identified:

• Monocytes: CD13+ CD11b+
  • Monocyte classical, non-classical, and intermediate populations based on CD14 and CD16 expression
• Granulocytes: CD13+ CD16+ CD14 lo CD11b+
• Eosinophils: CD13+ CD14– CD16– CD11b+

Table 8. List of Bio-Rad antibodies and viability dye used in the myeloid panel. Antibodies are colored according to the laser line: those in violet are excited by the 405 nm or violet laser, those in green by the 561 nm or yellow/green laser, and those in red by the 640 nm or red laser.  A647, Alexa Fluor 647; PE, phycoerythrin; SBV, StarBright Violet; SBY, StarBright Yellow.

Fig. 9. Myeloid panel data. Red blood cell lysed human peripheral blood was stained with the myeloid cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single cells prior to the gating strategy shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). A647, Alexa Fluor 647; PE, phycoerythrin; SBV, StarBright Violet; SBY, StarBright Yellow.