B Cell Activation on an Antigen-Coated Coverslip
JoVE Encyclopedia of Experiments
Immunology
0 views • 2:41 min • July 8th, 2025
B cell activation involves the recognition of antigens by B cell receptors, BCRs, on their surface to elicit an immune response.
To study B cell activation in vitro, begin with a coverslip containing antigens and anti-CD45R antibodies, immobilized over a polymeric material. The negatively-charged antigens and antibodies interact with positively-charged polymers on the coverslip through electrostatic interactions.
Overlay the coated coverslips with a medium containing B cells and incubate. The CD45R — the transmembrane protein of B cells — interacts with the immobilized antibodies, facilitating cell adhesion.
The BCRs on the adhered B cell interact with an antigen, forming an immune synapse — the coordination region for cell signaling and antigen internalization. This promotes cytoskeleton remodeling and initiates cell spreading, which increases the cell surface in contact with the antigens.
Further remodeling causes the movement of BCR-coupled antigens toward the center of the immune synapse. This cellular rearrangement causes the repositioning of the centrosome near the synaptic membrane that guides intracellular organelles, including lysosomes, toward the synapse.
The fusion of lysosomes with the membrane leads to the internalization of the antigen-BCR complexes. The internalized antigens are processed into peptide fragments, loaded onto class-II major histocompatibility complex or MHC molecules, and finally presented on activated B cells.
Prepare the antigen solution by adding anti-BCR and B220 in PBS to a final concentration of 10 micrograms per mL and 7.5 micrograms per mL respectively. Next, set the coverslip over a lid of 24-well plate covered with paraffin film, and add 40 microliters of antigen solution. Seal the plate, and then incubate at four degrees overnight.
To start the activation in coverslip, first, wash them with PBS, and let them dry for a few minutes. Next, add 150,000 B cells, and incubate at 37 degrees for the different time points. In both cases, when activating either with beads or antigen-coated slides, we recommend starting with the longest time point, and then proceeding to the shorter ones.
Overview
This article demonstrates an in vitro protocol for studying organelle dynamics in B cells during immune synapse formation. The method focuses on the sequence of events following antigen recognition by B cell receptors (BCRs), including cytoskeletal remodeling, immune synapse formation, lysosome movement, antigen internalization, and antigen presentation on MHC class II molecules.
Key Study Components
Area of Science
- Immunology
- Cell Biology
- Microscopy-based Cell Analysis
Background
- B cell activation is initiated by antigen recognition via BCRs.
- Immune synapse formation is critical for effective antigen processing and presentation.
- Cytoskeletal remodeling and organelle repositioning are essential for antigen internalization.
- Understanding these processes provides insight into adaptive immune responses.
Purpose of Study
- To visualize and analyze the dynamics of organelles, particularly lysosomes, during B cell immune synapse formation.
- To establish a reproducible in vitro system for studying B cell activation mechanisms.
- To detail the steps leading from antigen recognition to antigen presentation in B cells.
Methods Used
- Preparation of antigen-coated coverslips using anti-BCR and B220 antibodies in PBS.
- Immobilization of antigens and antibodies on coverslips via electrostatic interactions with a polymeric substrate.
- Incubation of B cells on coated coverslips to induce immune synapse formation.
- Observation of cytoskeletal remodeling, organelle movement, and antigen internalization at various time points.
Main Results
- B cells adhere to antigen-coated coverslips via CD45R-antibody interactions.
- BCR engagement with antigen triggers immune synapse formation and cytoskeletal changes.
- Centrosome and lysosome repositioning toward the synapse facilitates antigen internalization.
- Internalized antigens are processed and presented on MHC class II molecules on activated B cells.
Conclusions
- The described protocol enables detailed study of organelle dynamics during B cell activation.
- Immune synapse formation involves coordinated cytoskeletal and organelle rearrangements.
- This system provides a valuable platform for dissecting molecular mechanisms of antigen processing and presentation in B cells.
Frequently Asked Questions
Executive Industry Relevance
In vitro B cell activation on antigen-coated coverslips enables precise dissection of immune synapse formation and organelle dynamics, supporting early-stage immunology discovery. This system provides mechanistic clarity for antigen processing and presentation, informing target validation and de-risking in immunotherapeutic development. The approach enhances predictive confidence for downstream screening and translational research in adaptive immunity.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of B cell receptor signaling and immune synapse formation for functional target validation.
- Clarifies cytoskeletal and organelle remodeling mechanisms, reducing biological ambiguity in early discovery.
- Supports predictive confidence in antigen processing pathways relevant to immunotherapy targets.
Screening & Assay Development
- Establishes a reproducible in vitro system for standardized B cell activation assays.
- Facilitates quantitative measurement of antigen internalization and presentation for assay development.
- Prepares validated biological systems for downstream compound or antibody screening workflows.
Translational & Preclinical Research
- Aligns in vitro findings with disease-relevant immune mechanisms for translational continuity.
- Supports risk-adjusted advancement decisions by providing mechanistic insight into antigen presentation.
- Enables preclinical modeling of adaptive immune responses for immunomodulatory strategies.
Pipeline & Workflow Integration
This method integrates into the discovery continuum from early mechanistic studies to preclinical immunology research, supporting both target validation and assay readiness.
- Discovery Biology: Provides a platform for hypothesis testing of B cell activation and immune synapse dynamics.
- Screening: Delivers standardized, reproducible outputs for quantitative assessment of antigen processing.
- Analytics: Enables measurement of organelle movement, antigen internalization, and MHC presentation for comparative analysis.
- Translational Research: Bridges mechanistic discovery with preclinical immune function studies.
- Enterprise Reuse: Offers a reusable assay system for diverse immunology and cell biology programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic uncertainty in immune pathway studies.
- Operational Value: Standardizes B cell activation protocols for reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions and enhances capital efficiency in immunology portfolios.
- Portfolio Impact: Supports risk-adjusted prioritization of immunotherapeutic targets and modalities.
Implementation Considerations
- Requires expertise in immunology, cell culture, and microscopy-based analysis.
- Needs access to antigen-coating reagents, coverslip preparation tools, and imaging infrastructure.
- Demands cross-team standardization for assay reproducibility and data comparability.
- Adaptable to various antigen and antibody systems for broader application.
- Limited to in vitro modeling; in vivo validation may be required for translational claims.
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Last updated: 22 August 2026