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Published on: November 9, 2017
Multiparameter Intracellular Cytokine Staining
Patricia Lovelace1, Holden T Maecker2
1Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University, Fairchild Science Building, 299 Campus Drive, Stanford, CA, 94305-5124, USA.
Insights
This study reviews best practices for multicolor intracellular cytokine staining, a method to visualize cellular responses. It provides an optimized protocol to address technical and analytical challenges in flow cytometry.
Area of Science:
- Immunology
- Cellular Biology
- Biotechnology
Background:
- Intracellular cytokine staining visualizes cellular responses, particularly T-cell activation.
- It can be combined with markers for functional assays (e.g., CD107, CD154) and cell subset identification (e.g., T cells, NK cells, monocytes).
- Advances in multicolor flow cytometry allow for 12+ markers, presenting technical and analytical challenges and highlighting the need for standardization.
Purpose of the Study:
- To review best practices for antibody panel design in multicolor intracellular cytokine staining.
- To discuss procedural variables affecting intracellular cytokine staining.
- To present an optimized protocol with variations for specific markers and sample types.
Main Methods:
- Review of current literature and best practices in multicolor flow cytometry.
- Development and optimization of an intracellular cytokine staining protocol.
- Adaptation of the protocol for specific markers and sample types.
Main Results:
- Identification of key technical and analytical challenges in multicolor intracellular cytokine staining.
- Establishment of best practices for antibody panel design and procedural execution.
- Presentation of an optimized, adaptable protocol for intracellular cytokine analysis.
Conclusions:
- Standardization of multicolor intracellular cytokine staining protocols is crucial.
- The presented optimized protocol offers a reliable method for analyzing cellular responses.
- This work aims to improve the reproducibility and accuracy of flow cytometry-based immune monitoring.
Abstract:
Intracellular cytokine staining is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g., effector and memory T-cell compartments, NK cells, or monocytes. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 12 or more markers, creating some technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor intracellular cytokine staining, and present an optimized protocol with variations designed for use with specific markers and sample types.
