B-세포 연구

A multicolor immunophenotyping approach is well suited to the study of B cells, since B cells by nature require the use of more markers than, for example, T cells, to define the basic subsets present in most samples.


Backbone Markers

A wide variety of B-cell studies employs cell surface markers that define the major B-cell subsets, and these form the "backbone," or core, for panels to do more detailed analysis.

The key marker for B-cell panels is the lineage marker CD19, which is expressed by almost all cells belonging to the B-cell lineage. In the mouse, CD45R/B220 is traditionally used. Most panels also include surface-expressed IgD and IgM, since the BCR isotype provides information about the differentiation stage of the B cells.

To provide flexibility in panel design, BD offers all of these markers in a wide selection of formats.

Basic Panel

Depending on the type of sample that is being analyzed, different markers can be selected to define the specific B-cell subsets of interest. The example shown analyzes human peripheral blood using CD19, CD20, IgD, CD27, CD38, and CD24. With this six-color panel it is possible to identify transitional B cells (CD24high, CD38high), to discriminate between naïve and memory cells (naïve cells are CD27IgD+, memory cells CD27+IgD), and identify plasmablasts (CD38+).1

Deeper Phenotyping

Because the phenotype of some subsets is very similar to others, often a more extensive phenotyping panel is required. For example, a panel might distinguish the many B-cell developmental stages present in bone marrow B cells.

Adding markers can also provide a more fine-detail analysis, for example, defining memory cells or different types of plasma cells. Other molecules offer insight into the potential for cells to home and localize within the body (chemokine receptors, CD62L). Adding activation markers (CD69, CD25, CD80, CD86) can inform about which subsets are activated, and provide important clues about an individual's immune response.

Tools for Building Panels

Antigen Density. When adding markers to a panel, researchers can take advantage of antigen-density information to optimize antigen-fluor selection. In general, we recommend choosing bright fluorochromes for markers with low antigen density, and the less bright fluorochromes for highly expressed markers. To support researchers, BD is generating information about the antigen density of a number of markers on peripheral blood cells.

Bright Conjugates for Dim Markers. Certain B-cell populations are so rare, or defined by dimly expressed antigens, that achieving sufficient resolution is critical to obtaining good data in staining experiments. In particular for such rare or dim populations, the use of very bright fluorochromes helps improve resolution. A number of B-cell marker antibodies are available conjugated to very bright BD Horizon™ Brilliant Violet™ (BV) dyes, such as BD Horizon™ Brilliant Violet™ 421 (BV421), BD Horizon™ Brilliant Violet™ 510 (BV510), and BD Horizon™ Brilliant Violet™ 605 (BV605).

An Extended Panel for Defining Additional Subsets

In designing the panel used for the 10-color analysis experiment shown, information about antigen density helped optimize the fluorochrome distribution. Bright fluorochromes were chosen for the low-density antigen CD138 and to allow resolution of populations expressing different levels of IgD and CD38, and the extremely bright BV dyes were used to obtain optimal detection of CD27 and IgM.

Analysis using this panel provided the additional information about Ig subclass expression, allowing identification of both IgG+ and IgG post-class-switched memory cells (IgMIgDCD38CD27+/dim), and the clear discrimination of plasma cells (IgMIgDCD20CD38m++).

Six-color analysis of human peripheral blood cells
Six-color analysis of human peripheral blood cells

Human peripheral blood mononuclear cells (PBMCs) were stained with the following fluorescent antibodies: CD19 PerCP-Cy™5.5, CD20 APC-H7, IgD FITC, CD27 PE-Cy™7, CD38 APC, and CD24 PE, and analyzed using a BD FACSVerse™ flow cytometer.

Ten-color analysis of human peripheral blood cells
Ten-color analysis of human peripheral blood cells

Human PBMCs were stained with the following fluorescent antibodies: CD19 BD Horizon™ Brilliant Violet™ 711, CD20 Alexa Fluor® 700, IgD PE-Cy7, CD27 BV421, CD38 APC, CD24 BD Horizon™ PE-CF594, CD3 BD Horizon™ V500, CD138 PE, IgM BD Horizon™ Brilliant Violet™ 605 (BV605), and IgG FITC, and analyzed using a BD LSRFortessa™ flow cytometer.

Antigen density of B-cell markers on human PBMCs
Antigen density of B-cell markers on human PBMCs

The antigen density of B-cell markers on human PBMCs from three healthy donors was calculated using the BD Quantibrite™ bead method, and analysis on a BD LSRFortessa and a BD FACSVerse flow cytometer.


References

1. Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nature Rev Immunol. 2012;12:191-200.



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