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Detection of intracellular cytokines by flow cytometry
1Universitätskinderklinik im St. Josef-Hospital, Ruhruniversität Bochum, Germany.
Insights
This study presents a refined flow cytometry method for detecting intracellular cytokines like IFN-gamma, IL-2, and IL-4 in single cells. The technique enhances signal detection, enabling better characterization of cytokine-producing immune cells in various disease contexts.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cytokine production is not limited to a single cell type, necessitating methods to analyze cytokine expression at the single-cell level.
- Previous methods for detecting intracellular cytokines, such as indirect immunofluorescence microscopy, had limitations in sensitivity and throughput.
- Understanding the cytokine profiles of heterogeneous cell populations is crucial for deciphering immune responses in health and disease.
Purpose of the Study:
- To develop and validate a modified flow cytometry method for sensitive intracellular cytokine detection.
- To enable simultaneous analysis of intracellular cytokines and cell surface markers on single cells.
- To facilitate the characterization of cytokine-producing immune cell subsets in complex biological samples.
Main Methods:
- Intracellular cytokine staining using paraformaldehyde fixation, saponin permeabilization, and indirect immunofluorescence.
- Inclusion of monensin to block intracellular transport and accumulate cytokines in the Golgi complex, enhancing signal.
- Application of single and three-color flow cytometry for detecting Interferon-gamma (IFN-γ), Interleukin-2 (IL-2), and Interleukin-4 (IL-4).
Main Results:
- The modified method significantly increased the signal-to-noise ratio, allowing detection of weakly fluorescent cells, including IL-4 producers.
- Flow cytometry analysis showed high concordance with microscopic evaluation of cytokine production.
- Simultaneous detection of intracellular cytokines and cell surface markers identified distinct human T cell subpopulations with specific cytokine production patterns.
Conclusions:
- Intracellular cytokine detection by flow cytometry is a rapid, easy, and semiquantitative assay for analyzing heterogeneous cell populations.
- This method allows for the characterization of cytokine patterns in specific immune cell subsets without the need for cell sorting.
- The technique is valuable for studying the role of distinct immune cell subsets and their cytokine production in various disease states.
Abstract:
During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.