Detection of Intracellular Cytokines by Flow Cytometry
Yuzhi Yin1, Alyssa Mitson-Salazar1,2, Calman Prussin1
1National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.
Insights
Intracellular cytokine staining (ICCS) using flow cytometry is a powerful method to analyze cytokine expression in single cells. This technique allows for multiplex cytokine analysis and offers advantages over other single-cell methods.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Intracellular cytokine staining (ICCS) is a key technique for analyzing cytokine expression at the single-cell level.
- Advances in polychromatic flow cytometry have significantly improved the sophistication and capabilities of ICCS.
- ICCS enables the simultaneous measurement of multiple cytokines and other cellular analytes.
Purpose of the Study:
- To describe the methodology for intracellular cytokine staining (ICCS) of in vitro-stimulated and fixed cells.
- To highlight the advantages of ICCS over other single-cell analysis techniques.
- To provide methods for cell activation, fixation, and surface staining for comprehensive ICCS analysis.
Main Methods:
- Utilizing fluorescently labeled monoclonal antibodies (MAbs) for detection via flow cytometry.
- Employing polychromatic flow cytometry for simultaneous measurement of multiple cytokines and analytes.
- Implementing in vitro cell activation (e.g., PMA/ionomycin, antigens), cell fixation, and cell surface staining protocols.
Main Results:
- ICCS allows for the detection of complex cytokine phenotypes within single cells.
- Multiple cytokines can be measured concurrently with other analytes like transcription factors and proliferation dyes.
- High-throughput analysis is achievable, offering significant advantages over methods like ELISPOT or T cell cloning.
Conclusions:
- ICCS is a premier technique for single-cell cytokine expression analysis.
- The described methods facilitate robust ICCS for studying immune responses.
- ICCS provides a high-throughput, multiplexed approach for detailed cellular analysis.
Abstract:
Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.
