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Updated: Aug 8, 2026
Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Simultaneous detection of cytokine and immunophenotype at the single cell level by immunoenzymatic double staining
D M Behringer1, B Sunderer, U Andersson
1Department of Haematology/Oncology, University of Freiburg, Germany.
Insights
This study developed a new method to detect cytokines and cell surface markers simultaneously in single cells. This technique uses standard lab equipment and aids in precise cell identification for research and diagnostics.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Simultaneous detection of cytokine production and immunophenotype in single cells is crucial for understanding cellular function.
- Existing methods often lack efficiency or require specialized equipment, limiting their widespread application.
Purpose of the Study:
- To establish a versatile immunoenzymatic method for simultaneous single-cell detection of cytokines and immunophenotypes.
- To optimize cell preparation, fixation, and staining protocols for enhanced discrimination.
Main Methods:
- Utilized saponin (0.1%) for cell permeabilization and glutaraldehyde (0.04%) for fixation.
- Employed sequential immunoperoxidase labeling with diaminobenzidine for cytokines and 4-chloronaphthol for immunophenotypes.
- Validated the use of primary monoclonal antibodies from the same species without compromising sensitivity or specificity.
Main Results:
- Achieved precise identification of double-labeled cells through distinct staining patterns ('Golgi staining' for cytokines, 'ring-like' for immunophenotype).
- Demonstrated the method's effectiveness with standard laboratory equipment.
- Confirmed no loss of sensitivity or specificity when using antibodies from the same species.
Conclusions:
- The developed method enables simultaneous analysis of morphology, cytokine production, and immunophenotype at the single-cell level.
- This technique offers a valuable tool for immunological research and has potential applications in histopathological diagnostics by incorporating cytokine profiles.
Abstract:
The goal of this study was to establish a generally applicable immunoenzymatic method for the simultaneous detection of cytokine and immunophenotype at the single cell level. Evaluating various cell preparations and staining protocols, we found that permeabilization by saponin (0.1%) is very efficient, in combination with glutaraldehyde (0.04%) as fixative. Among various staining procedures, sequential immunoperoxidase labelling of the cytokine by use of diaminobenzidine, and detection of the immunophenotype by use of 4-chloronaphthol proved most discriminative. The typical localization of the cytokine reaction product ('Golgi staining') within the cell, and the 'ring-like' staining for the immunophenotype on the cell surface, allowed precise identification of double-labelled cells. Primary monoclonal antibodies from the same species could be used without loss of sensitivity and specificity for either or both antigens. This method thus provides the opportunity to study morphology, cytokine and immunophenotype simultaneously at the single cell level with standard equipment. Its application for the analysis of tissue samples is in progress, and may allow us to incorporate the cytokine-type as a new parameter in histopathological diagnostics.
