Related Experiment Video
Updated: Jul 3, 2026
Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Standardization and optimization of multiparameter intracellular cytokine staining
Laurel Nomura1, Vernon C Maino, Holden T Maecker
1BD Biosciences, 2350 Qume Dr., San Jose, California 95131, USA. laurel_nomura@bd.com
Insights
Optimizing multicolor flow cytometry for intracellular cytokine staining (ICS) is crucial for vaccine research. Standardizing protocols and using tools like lyoplates can reduce variability in T-cell analysis across labs.
Area of Science:
- Immunology
- Cellular Biology
- Biotechnology
Background:
- Intracellular cytokine staining (ICS) is essential for quantifying antigen-specific T cells, particularly for assessing vaccine immunogenicity (e.g., IFNgamma, IL-2 production).
- Increasing availability of fluorochromes and advanced flow cytometers complicates multicolor ICS panel design and raises concerns about inter-instrument variability.
Purpose of the Study:
- To review challenges and solutions for multiparameter (>four color) flow cytometry in intracellular staining.
- To address issues in antibody-fluorochrome matching, panel optimization, instrument setup, controls, sample management, and data analysis.
- To highlight the utility of lyophilized reagents (lyoplates) for reducing variability and processing time in multi-site studies.
Main Methods:
- Focus on multiparameter flow cytometry techniques for intracellular cytokine detection.
- Discussion of antibody-fluorochrome selection and optimization strategies.
- Examination of standardization methods, including the use of lyoplates and multi-laboratory studies.
Main Results:
- Identified challenges in selecting optimal fluorochrome combinations for multicolor ICS.
- Demonstrated the potential of lyoplates to decrease experiment-to-experiment variability and processing time.
- Presented data from multi-laboratory standardization studies highlighting sources of variability in flow cytometry data acquisition.
Conclusions:
- Standardization of ICS protocols and reagent use is critical for reliable vaccine immunogenicity assessment.
- Multiparameter flow cytometry requires careful optimization of reagents, instruments, and data analysis to ensure consistency.
- Lyoplates offer a promising approach to enhance reproducibility in multi-center T-cell assays.
Abstract:
Intracellular cytokine staining (ICS) is a common method for rapid quantitation of cytokine-producing antigen-specific T cells. T cell production of IFNgamma in particular, and more recently IL-2 as well, is often taken as a measure of vaccine immunogenicity in experimental vaccine trials. As more fluorochromes become available for use in ICS and other applications detecting intracellular markers, the selection of optimal fluorochrome combinations becomes correspondingly more complicated. Additionally, as more sophisticated flow cytometers become available, more attention is being paid to potential result variability from one instrument to another. This review summarizes an oral presentation given at MASIR 2008, January 30-Feb 1, 2008, in La Plagne, France. We focus on issues associated with multiparameter (>four color) flow cytometry, including matching antibody specificities with available fluorochromes and techniques to optimize fluorochrome combinations. We examine issues specific to intracellular staining as well as broader topics such as instrument setup, experimental controls, sample management, and analysis of multiparameter data sets. Particular emphasis is placed on the use of lyophilized cells, antibodies, beads, peptides, etc. (collectively known as "lyoplates"), which can decrease experiment-to-experiment variability as well as processing time. Most clinical trials compile results from multiple testing sites, using data that was acquired on-site in each location. We present data from two different ongoing multi-laboratory standardization studies, one involving 15 laboratories and one involving nine. We identify issues of variability and, where possible, offer solutions.
