B-Cell Research

The development, activation, and differentiation of B cells are accompanied by a number of changes in intracellular molecules, including transcription factors, phosphosignaling proteins, and cytokines.

BD offers solutions that uniquely enable analysis of these intracellular molecules, to support researchers in deciphering the interconnected pathways regulating B-cell biology.

Basic principles of intracellular staining - Main

Basic principles of intracellular staining

Cells are fixed and permeabilized (symbolized by dashed line membrane), stained, and then analyzed by flow cytometry. For studies of secreted proteins, cells are first treated with a protein transport inhibitor to allow accumulation of the target protein inside the cell.


Bcl-6 expression in mouse lymph node cells
Bcl-6 expression in mouse lymph node cells

Mouse lymph node cells were stained with anti CD45R/B220, CD4, IgD, and CD95, fixed and permeabilized using the BD Pharmingen™ transcription factor buffer set, and stained with anti-Bcl-6 Alexa Fluor® 488. Flow cytometry was performed using a BD™ LSR II system. Bcl-6 expression was observed in germinal center B cells, identified using the CD4B200+IgDloCD95hi phenotype.

Intracellular Detection

Multicolor flow cytometry is a powerful technique for the analysis of intracellular molecules. Simultaneous analysis with markers for specific cell subsets can reveal information about the differentiation and activation state of distinct populations of B cells. While techniques for cell surface staining are relatively standard, optimal staining for intracellular markers often depends on the biology of the target protein.

Specialized Buffers and Antibodies

To facilitate the detection of these intracellular molecules by flow cytometry, BD has developed monoclonal antibodies, specialized buffer systems, and kits that are optimized for detection of specific types of intracellular targets. Flow cytometry has several advantages over commonly used methods such as Western blot: multiple parameters can be measured simultaneously from heterogeneous samples such as blood. This conserves precious samples and provides detailed results at the cellular level.

Phospho-mTOR staining of human cells
Phospho-mTOR staining of human cells

Human peripheral B lymphocytes were stimulated with CpG ODN2395, then fixed with BD Cytofix™ fixation butter, permeabilized using BD Phosflow™ perm buffer III, and stained with anti-mTOR (pS2448) PE or a matching isotype control, and anti-CD20 Alexa Fluor® 647. Cells were analyzed using a BD FACSCanto™ II system.

Detection of Transcription Factors

The BD Pharmingen™ transcription factor buffer permeabilizes the cells sufficiently to allow the exposure of intranuclear epitopes, while still being gentle enough to allow the detection of most cell surface proteins. It can be used alone or in combination with cell surface markers and cytokines for an improved detection of a number of different transcription factors.

BD offers several B-cell–relevant, flow-validated transcription factor antibodies, including unique specificities. Some, such as Oct-2 and Pax-5, are expressed through the follicular or germinal center (GC) stages. Bcl-6 characterizes proliferating GC B cells, while others, such as Blimp-1 and XPB-1s, are typical for plasma cells.

Blimp-1 staining in activated mouse splenocytes
Blimp-1 staining in activated mouse splenocytes

B6 mouse splenocytes activated with LPS for 3 days were analyzed using the BD Pharmingen transcription factor buffer set, anti-CD45R/B220 PE, and either anti-Blimp-1 Alexa Fluor® 647 or a matching isotype control. Flow cytometry was performed using a BD FACSCanto II system.

Detection of Phosphoproteins

BD Phosflow™ antibodies are monoclonal phosphoepitope– specific antibodies validated for flow cytometric detection. The recommended permeabilization buffer for most BD Phosflow antibodies is BD Phosflow™ perm buffer III, but alternative permeabilization buffers also are available to meet particular experimental needs.

A number of BD Phosflow antibody specificities are available for analysis of signaling pathways involved in B-cell development and activation, including phospho-Akt, phospho-Btk, phospho-Syk, phospho-BLNK, phospho-CD20/ BL-CAM, phospho-IKKγ, phospho-NFκB, and phospho-mTOR.

XBP-1s expression in CpG-stimulated human PBMCs
XBP-1s expression in CpG-stimulated human PBMCs

CpG-stimulated human PBMCs were incubated with BD Horizon™ fixable viability stain 450, fixed and permeabilized using the BD Pharmingen transcription factor buffer set, and stained with anti-CD20 PerCP-Cy™5.5, and either anti-XBP-1s PE or a matching isotype control. Flow cytometry was performed using a BD FACSCanto II system.

Detection of Cytokines

Using a protein transport inhibitor to block secretion, researchers can detect cytokines in the cell in which they are being produced. This makes it possible to easily determine if the cytokine production by an activated cell population is the result of a few cells producing large amounts of cytokine or a large population of cells producing small quantities of cytokine per cell.

BD’s well established kits, buffer systems, and protocols for intracellular cytokine staining include BD Cytofix/ Cytoperm™ fixation/permeabilization solution, which is suitable for staining most cytokines and cell surface markers. A wide selection of direct conjugates is available, covering cytokines often used to distinguish Bregs, and those secreted by Be-1 and Be-2 cells.



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