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Published on: July 23, 2020
Analysis of B Cell Receptor-Mediated Antigen Extraction by B Lymphocytes from Plasma Membrane Sheets Using Confocal
Abhijit Ashok Ambegaonkar1, Haewon Sohn2
1Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA. abhijit.ambegaonkar@nih.gov.
Insights
This study details a confocal microscopy protocol for observing B cell receptor (BCR)-mediated antigen internalization. The method efficiently analyzes rare cell types, advancing endocytosis research.
Area of Science:
- Cell biology
- Immunology
- Microscopy
Background:
- Receptor-mediated endocytosis is crucial for cellular processes.
- Confocal microscopy offers high-resolution insights into endocytosis.
- Investigating B cell receptor (BCR) signaling requires specific methodologies.
Purpose of the Study:
- To present a detailed protocol for analyzing B cell receptor (BCR)-mediated antigen internalization using confocal microscopy.
- To provide a method for studying receptor-mediated endocytosis in various cell types, particularly rare ones.
- To enable quantitative analysis of antigen uptake via BCR.
Main Methods:
- Preparation of plasma membrane sheets (PMS) for localized antigen binding.
- Activation of B cells on PMS with fluorescently tagged antigens.
- High-resolution confocal imaging and computational image analysis for internalization assessment.
Main Results:
- The protocol allows for the visualization and quantification of antigen internalization via BCR.
- The method is effective even with limited numbers of rare cell types.
- Successful application demonstrated for B cells, with potential for other cell types.
Conclusions:
- This confocal microscopy protocol provides a robust method for studying BCR-mediated antigen internalization.
- The technique is valuable for endocytosis research, especially in scenarios with limited cell availability.
- The protocol can be adapted for investigating receptor-mediated endocytosis in diverse cellular contexts.
Abstract:
High-resolution confocal imaging has provided new insights in the process of receptor-mediated endocytosis in variety of cell types. We describe here the protocol for investigating B cell receptor (BCR)-mediated internalization of membrane bound antigens using confocal microscopy. We describe the method to prepare plasma membrane sheets (PMS) in a small area, bind fluorescently tagged antigens to the PMS and activate B cells on the PMS. We also describe the method for analyzing antigen internalization using confocal microscopy and computational image analysis. This protocol is useful for the study of antigen internalization by B cells and can be applied for studying receptor-mediated endocytosis in other cells as well. The setup we describe here is especially useful for studying rare cell types when the number of cells available is limiting.
