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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.

Methods in Molecular Biology (Clifton, N.J.)
|October 27, 2017
PubMed
Summary
This summary is machine-generated.

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.

Keywords:
AIDS vaccine researchAntigen-specificFixationIntracellular stainingMulticolorPermeabilizationPolychromaticT cells

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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.