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Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues
Hans Christian Probst1,2, Patrizia Stoitzner3, Lukas Amon4
1Institute of Immunology, University Medical Center Mainz, Mainz, Germany.
Insights
This article offers detailed protocols for isolating and analyzing dendritic cells (DCs) from various tissues. These guidelines enhance the study of immune responses in both health and disease.
Area of Science:
- Immunology
- Cell Biology
Background:
- Dendritic cells (DCs) are crucial immune sentinels found in most mammalian organs.
- Their rarity necessitates specialized extraction protocols for each tissue type.
Purpose of the Study:
- To provide comprehensive, state-of-the-art protocols for dendritic cell research.
- To facilitate the preparation, analysis, and characterization of DCs from diverse tissues.
Main Methods:
- Detailed protocols for single-cell suspension preparation from mouse nonlymphoid tissues (skin, intestine, lung, etc.).
- Guidelines for multiplex flow cytometry analysis and DC subset discrimination.
- Methods for fluorescence microscopy and functional characterization.
Main Results:
- Established protocols for isolating and analyzing dendritic cells from various lymphoid and nonlymphoid tissues.
- Methods ensure proper discrimination of dendritic cell subsets from other myeloid cells.
Conclusions:
- This collection serves as an essential resource for immunologists studying dendritic cells.
- The protocols advance the understanding of dendritic cell roles in healthy and diseased states.
Abstract:
This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.
