B-Cell Research

Instrument setup for multicolor experiments using antibody panels of more than four colors can be time consuming.

When a panel of five markers is measured on two lasers, the fluorochromes that are typically used exhibit spillover into other fluorescence channels, thus requiring compensation. Researchers who have access to an instrument with four or five lasers can use this capability to their advantage, to simplify setup and minimize the need for compensation.

Five-color B-cell panel: minimal compensation
Five-color B-cell panel: minimal compensation

Human peripheral blood mononuclear cells (PBMCs) were stained with the following fluorescent antibodies: CD19 BUV395, CD27 BV421, IgD FITC, CD38 PE-CF594, and IgM APC. Samples were acquired using a 5-laser BD LSRFortessa™ flow cytometry system.

Five-laser instruments are often equipped with a 355-nm UV laser, which is typically used for side population analysis, calcium measurement using indo-1, or viability staining. To allow users to take full advantage of this equipment for phenotyping experiments, BD has developed a new UV-excited polymer-based dye, BD Horizon™ Brilliant Ultraviolet™ 395 (BUV395). Incorporating antibodies conjugated to BUV395 into a panel in which each fluorochrome is excited by a different laser results in minimal or no spillover between them.

This is illustrated by an example of a 5-color panel discriminating naive and memory B cells. Using this panel, only minimal setup and virtually no compensation were needed.

Combining Measurements to Maximize Results

Advances in buffer systems and methodologies now make it easier to simultaneously measure both surface and intracellular markers, and help researchers move toward information-rich, combined measurements. For example, several cell types in a heterogeneous sample of differentiating B cells can be monitored using a combination of cell surface and intracellular markers, to efficiently get more relevant data out of each sample.

The availability of antibodies to both B-cell surface markers and specific transcription factors in many different conjugates, including new BD Horizon™ dyes, adds flexibility when designing multicolor experiments.

Analysis of Pax-5 expression in mouse splenic B-cell subpopulations
Analysis of Pax-5 expression in mouse splenic B-cell subpopulations

C57BL/6 mouse splenocytes were stained with the following fluorescent antibodies: CD11b Alexa Fluor® 700, IgM FITC, CD19 BUV395, CD45R/B220 APC, CD93 PE, IgD BD Horizon™ Brilliant Violet™ 605 (BV605), CD21 PerCP Cy™5.5, CD23 BD Horizon™ Brilliant Violet™ 421 (BV421), and CD3 BD Horizon™ Brilliant Violet™ 711 (BV711). Following surface staining, cells were fixed and permeabilized using the BD Pharmingen™ transcription factor buffer set, intracellularly stained with BD Horizon™ Brilliant Violet™ 510 (BV510) anti Pax-5, and analyzed using a special order BD LSRFortessa™ X-20 system.

Monitoring Pax-5 in Splenic B-cell Subpopulations

In the example shown, nine cell surface markers were used to analyze B-cell subsets in mouse spleen. They allowed discrimination between the different developmental phases present in this tissue. Follicular B I and II and marginal cell subsets (Marginal Zone and Marginal Zone Progenitor) were identified based on expression of IgM, IgD, CD21, and CD23. The different levels of IgM and CD23 expression helped distinguish the stages of transitional subsets (T1, T2, and T3).

Measurement of the expression of Pax-5 in the different subsets revealed differential Pax-5 expression in follicular and marginal zone B cells.

Alexa Fluor® is a registered trademark of Life Technologies Corporation.

Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA.