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Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
Published on: July 4, 2018
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Protocol to desensitize human and murine mast cells after polyclonal IgE sensitization
Celia López-Sanz1, Elisa Sánchez-Martínez1, Rodrigo Jiménez-Saiz2
1Department of Immunology, Instituto de Investigación Sanitaria Hospital Universitario de La Princesa (IIS-Princesa), Universidad Autónoma de Madrid (UAM), Madrid, Spain.
STAR Protocols
|October 12, 2022
Summary
This protocol details how to desensitize mast cells (MCs) for allergy research. Desensitized MCs can then be used in various downstream applications, including co-cultures and imaging studies.
Area of Science:
- Immunology
- Cell Biology
Background:
- Mast cells (MCs) play a crucial role in allergic responses.
- IgE-mediated activation of mast cells leads to degranulation and mediator release.
- A method to transiently reduce mast cell responsiveness is needed for specific experimental setups.
Purpose of the Study:
- To provide a detailed protocol for desensitizing human and murine mast cells (MCs) following IgE sensitization.
- To outline methods for assessing mast cell degranulation after desensitization.
Main Methods:
- Polyclonal IgE sensitization of mast cells.
- Desensitization procedure for mast cells.
- Assessment of mast cell degranulation via flow cytometry (measuring CD63 and CD107a expression) and β-hexosaminidase activity assay.
Main Results:
- Successful desensitization of both human and murine mast cells was achieved.
- Established methods for quantifying degranulation in desensitized mast cells.
Conclusions:
- Desensitized mast cells are viable for use in subsequent experiments.
- This protocol enables the use of mast cells in co-culture, immunofluorescence, live imaging, and omics approaches.
