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Updated: Aug 10, 2026
Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry
Published on: March 5, 2019
Multiple color immunofluorescence for cytokine detection at the single-cell level
Matthew J Loza1, Jeffrey S Faust, Bice Perussia
1Bice Perussia, Jefferson Medical College, Kimmel Cancer Institute, Philadelphia, PA 19107, USA.
Insights
This study introduces a new immunofluorescence flow cytometry method to detect intracellular cytokines in mixed immune cells. This technique precisely identifies cytokine-producing cells and their production levels within complex cell populations.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cytokine detection and producer cell identification are crucial for understanding immune and inflammatory responses.
- Existing methods like ELISA and RT-PCR have limitations in resolving specific cell subsets and require large cell numbers.
Purpose of the Study:
- To present a novel immunofluorescence-based flow cytometry method for intracellular cytokine detection.
- To enable precise identification and quantification of cytokine-producing cells within mixed leukocyte populations.
Main Methods:
- Utilized immunofluorescence and flow cytometry to detect intracellular cytokine accumulation.
- Applied the method to natural killer (NK) cells as a model, demonstrating its applicability to other cell types.
Main Results:
- The method allows for the identification of cytokine-producing cells within mixed populations.
- Enables quantitative comparison of cytokine production levels across different cell subsets.
- Facilitates the detection of simultaneous cytokine production by single cells.
Conclusions:
- The described immunofluorescence flow cytometry method offers a powerful tool for analyzing cytokine expression in complex cellular environments.
- It is particularly valuable for studying rare immune cell subsets and simultaneous cytokine production.
Abstract:
Detection of cytokines and identification of the producer cells are essential to define the interplay and the role of distinct leukocyte subsets in the development of immune and inflammatory responses. Several methods used to study cytokine expression are based on detection of the encoding mRNA (Northern blot, RNase protection assay, RT-PCR) or of protein in the supernatant from stimulated cells (ELISA, RIA, ELISPOT). These are simple and useful, but have limitations related to the need of using purified cell populations to precisely define the effector cells, and exception made for RT-PCR and ELISPOT assays, the requirement for relatively large numbers of cells for sufficient resolution. Here we present a method based on immunofluorescence (flow cytofluorimetry) to detect intracellular accumulation of cytokines in mixed leukocyte populations. It has the distinct advantage of: 1. identifying producing cells in mixed cell populations, 2. comparing quantitatively the levels of production within cells in a given population and among different cell subsets, and 3. defining simultaneous production of distinct cytokines by a single cell type. The detailed description/discussion of the method uses natural killer (NK) cells as an example, but this method can be applied to the study of other cell types, and is of special interest/use when analyzing subsets present in very low proportion.
