Murine No Compensation Panels

Identification of the four major cell types is shown below. Whole blood is required for this panel; peripheral blood mononuclear cells (PBMCs) cannot be used as the preparation of PBMCs removes granulocytes. B cells can also be identified using CD45R/B220 antibody rather than CD19. Table 1 lists the panel antibodies; Figure 2 shows sample data.

Markers used to identify target cell populations:

• T cells: CD3+ CD11b– CD19– Ly6G–
• B cells: CD3– CD11b– CD19+ Ly6G–
• Monocytes: CD3– CD11b+ CD19– Ly6G–
• Granulocytes: CD3– CD11b+ CD19– Ly6G+

Table 1. List of Bio-Rad antibodies and viability dye used in the T, B, and myeloid 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. 2. T, B, and myeloid cell panel data. Red cell lysed mouse 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 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.

This basic panel will identify T helper and cytotoxic T cell subsets. By including CD44 and CD62L markers, it is also possible to distinguish naïve (CD62L+CD44–), memory (CD62L– CD44–), and effector (CD62L– CD44^mid) T cells for both the helper and cytotoxic T cell populations. Table 2 lists the panel antibodies; Figure 3 shows sample data.

Markers used to identify target cell populations:

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

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

 

Fig. 3. T cell panel data. Splenocytes from C57BL/6 mice were stained with the T cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single cells prior to the gating shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). SBB, StarBright Blue; SBUV, StarBright UltraViolet; SBV, StarBright Violet; SBY, StarBright Yellow.

This is a generalized staining panel used to identify naïve and memory B cells; it will only give an indication of the amount of naïve and memory B cells. This limitation arises because surface markers CD80 and CD273 (PD-L2) exhibit variable expression, making definitive subtype identification unfeasible. For more accurate characterization of these and other populations, such as marginal zone B cells, additional markers—including CD38, CD62L, CD73, CD21, and CD23—may be incorporated, depending on tissue type. Table 3 lists the panel antibodies; Figure 4 shows sample data.

Markers used to identify target cell populations:

• Naïve B cells: CD19+ CD45R+ CD80– CD273–
• Memory B cells: CD19+ CD45R+ CD80+ CD273+

Table 3. List of Bio-Rad antibodies and viability dye used in the 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; SBR, StarBright Red; SBV, StarBright Violet; SBY, StarBright Yellow.

 

Fig. 4. B cell panel data. Red cell lysed mouse blood was stained with the B cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single lymphocytes prior to the gating shown. Data were analyzed using FCS Express 7 (De Novo Software by Dotmatics). FITC, fluorescein isothiocyanate; SBR, StarBright Red; SBV, StarBright Violet; SBY, StarBright Yellow.

This panel enables the identification of NK and NKT cells and their maturation status. The pan NK/NKT marker CD161 used in this panel is not expressed in certain mouse strains, such as BALB/c. For these strains, an antibody against CD335/NKp46 can be used instead. Table 4 lists the panel antibodies; Figure 5 shows sample data.

Markers used to identify target cell populations:

• Immature NK cells: CD45+ CD3– CD161+ CD49b–
• Mature NK cells: CD45+ CD3– CD161+ CD49b+
• NKT cells: CD45+ CD3+ CD161+ CD49b+

Table 4. List of Bio-Rad antibodies and viability dye used in the NK and NKT 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. 5. NK and NKT cell panel data. Splenocytes from C57BL/6 mice were stained with the NK and NKT cell panel and data were acquired on a ZE5 Cell Analyzer. Cells were gated on live, single cells prior to the gating 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.