Flow Cytometry-Based Cell Sorting for Different Circulatory B Cell Populations
JoVE Encyclopedia of Experiments
Immunology
0 views • 3:01 min • July 8th, 2025
Based on their cell surface receptors, human circulating B cells are classified as CD19-positive naïve B cells, CD19 and CD27-positive memory B cells, and CD19, CD27, and CD38-positive plasmablast plasma cells.
To sort these cell populations, take a purified B cell suspension. Incubate with human immunoglobulin G antibodies. The antibodies block the B cells' Fc receptors, preventing non-specific antibody binding.
Add fluorescently-labeled antibodies targeting CD19, CD27, and CD38.
The anti-CD19 antibodies bind to the CD19 receptors ubiquitously expressed on B cells.
The anti-CD27 antibodies bind to the CD27 receptors expressed on memory B cells and plasmablast plasma cells.
The anti-CD38 antibodies bind to the CD38 receptors expressed on plasmablast plasma cells.
Add a fluorescent DNA-binding dye as a dead cell marker.
Dead cells exhibit a loss of membrane integrity, allowing the cellular entry of the dye and binding to the double-stranded DNA; live cells remain unstained.
Centrifuge to remove unbound antibodies and dyes and resuspend the cells in FACS buffer. Filter the cells through a strainer to obtain a homogeneous suspension and eliminate clumps.
Using a flow cytometer, sort the dead cells from the live ones. Within the live cells, CD19, CD27, and CD38 expression distinguish naïve B cells, memory B cells, and plasmablast plasma cells.
After performing non-specific blocking, add 1 microgram of each selection antibody per 1 x 106 cells to the tube with gentle mixing for a 30-minute incubation on ice. During the last five minutes of the incubation, add 5 microliters of 7-AAD to the cells. Then, add 2 milliliters of fresh PBS to the cells, and vortex before centrifuging.
Resuspend the pellet in fresh sorting buffer at a 1 to 5 x 107 cells per milliliter concentration, and filter the cells through a 40-micron cell strainer into a new 5-milliliter polystyrene tube. Then, sort the cells by flow cytometry into three 15-milliliter tubes containing 5 milliliters of fresh RPMI medium to collect the naive B cells, memory B cells, and plasmablast plasma cells.
Overview
This article details the classification and sorting of human circulating B cells based on their cell surface receptors. It outlines the methodology for isolating naïve B cells, memory B cells, and plasmablast plasma cells using flow cytometry.
Key Study Components
Area of Science
- Immunology
- Cell Biology
- Flow Cytometry
Background
- Human circulating B cells can be categorized into distinct populations.
- Cell surface receptors CD19, CD27, and CD38 are critical for this classification.
- Flow cytometry is a powerful tool for sorting and analyzing these cell types.
- Understanding B cell populations is essential for immunological research and therapeutic applications.
Purpose of Study
- To provide a detailed protocol for the sorting of B cell populations.
- To enhance the understanding of B cell functionality and differentiation.
- To facilitate research in immunology and related fields.
Methods Used
- Purification of B cell suspension and incubation with antibodies.
- Use of fluorescently-labeled antibodies to identify specific B cell populations.
- Application of a dead cell marker to distinguish live cells.
- Flow cytometry for sorting and analysis of B cell populations.
Main Results
- Successful identification and sorting of naïve B cells, memory B cells, and plasmablast plasma cells.
- Demonstrated effectiveness of fluorescent antibodies in distinguishing B cell types.
- Established a reliable protocol for future immunological studies.
- Highlighted the importance of cell viability markers in flow cytometry.
Conclusions
- The methodology presented is effective for B cell sorting and analysis.
- Understanding B cell populations can lead to advancements in immunotherapy.
- This study provides a foundation for further research in B cell biology.
Frequently Asked Questions
Executive Industry Relevance
Flow cytometry-based sorting of circulating B cell subpopulations enables precise isolation of naïve, memory, and plasmablast plasma cells from human peripheral blood. This capability supports target validation in immunology by providing disease-relevant systems for mechanistic de-risking of humoral immunity pathways. The method enhances predictive confidence in early discovery by delivering purified, functionally defined cell populations for downstream assay development and screening.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of B cell differentiation pathways and functional validation of antibody-secreting cell targets.
- Operational Value: Provides purified naïve, memory, and plasmablast plasma cell populations for target de-risking.
Screening & Assay Development
- Scientific Value: Generates standardized, viable B cell subsets for assay preparation and compound screening.
- Operational Value: Ensures reproducibility through dead cell exclusion and consistent surface marker gating.
Translational & Preclinical Research
- Scientific Value: Supports disease-relevant systems by isolating clinically relevant B cell subsets from peripheral blood.
- Operational Value: Enables continuity from discovery to preclinical validation using functionally defined cell populations.
Pipeline & Workflow Integration
The method integrates into the discovery continuum by supplying validated B cell populations for hypothesis testing, assay readiness, and analytical readouts that inform lead identification decisions.
- Discovery Biology: Supports hypothesis testing and pathway clarification through isolation of distinct B cell functional states.
- Screening: Delivers assay-ready, homogeneous cell suspensions with quantitative viability and subset purity metrics.
- Analytics: Enables comparison of cellular responses across subsets using flow cytometry-based quantitative readouts.
- Translational Research: Connects to preclinical continuity by providing peripheral blood-derived B cells reflective of in vivo immunity.
- Enterprise Reuse: Establishes a reusable sorting workflow for multiple immunology targets and therapeutic modalities.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through isolation of functionally defined B cell populations.
- Operational Value: Standardization and reproducibility via dead cell exclusion and consistent antibody staining.
- Strategic Value: Improved go/no-go decisions by reducing biological ambiguity in humoral immunity assays.
- Portfolio Impact: Risk-adjusted prioritization of B cell-targeted therapeutics based on subset-specific functionality.
Implementation Considerations
- Requires expertise in flow cytometry, antibody panel design, and B cell immunology.
- Dependent on fluorescence-activated cell sorter and compatible detection channels for CD19, CD27, CD38, and viability dyes.
- Necessitates standardization of antibody titration, incubation conditions, and gating strategies across users.
- Requires adaptation of antibody panels when applying to non-human or disease-altered B cell samples.
- Limited by sample quality and viability; necessitates fresh peripheral blood processing to maintain subset integrity.
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Last updated: 29 August 2026