A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid
Yen-Rei A Yu1, Emily G O'Koren2, Danielle F Hotten3
1Department of Medicine, Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Insights
This study presents a comprehensive flow cytometry protocol for analyzing immune cells in various non-lymphoid tissues. The method accurately quantifies 11 immune cell types, aiding in understanding immune responses and identifying tumor-associated macrophages.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry is crucial for immune cell analysis in non-lymphoid tissues.
- Existing methods lack comprehensiveness and broad applicability.
- A standardized protocol is needed for detailed immune cell profiling.
Purpose of the Study:
- To develop a comprehensive and widely applicable flow cytometry protocol for non-lymphoid tissues.
- To enable simultaneous identification and quantification of major immune cell types.
- To characterize immune cell populations in normal and inflamed tissues, including tumor microenvironments.
Main Methods:
- Developed a protocol including tissue preparation and a 10-fluorochrome staining panel.
- Implemented a standardized gating strategy for immune cell identification.
- Applied the protocol to various normal and inflamed non-lymphoid tissues and tumor models.
Main Results:
- The protocol minimizes cell loss and reliably distinguishes macrophages from dendritic cells (DCs).
- Accurately quantifies 11 distinct immune cell types across multiple tissues (lung, heart, liver, kidney, intestine, skin, eyes, mammary gland).
- Identified distinct tumor-associated macrophage phenotypes specific to individual tumor cell lines.
Conclusions:
- This protocol offers a valuable tool for examining immune cell repertoires in diverse tissues and conditions.
- It facilitates the study of immune responses and the characterization of immune infiltrates.
- The protocol can be expanded for detailed phenotyping and identification of additional immune cell subsets.
Abstract:
Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.
