Intracellular staining and detection of cytokines by fluorescence-activated flow cytometry

Area of Science:

• Immunology and Flow Cytometry
• Cellular and Molecular Medicine

Background:

• Measuring immune reactivity has been revolutionized by detecting cytokines within producing cells.
• Intracellular cytokine staining is the sole immunological method enabling simultaneous determination of antigen-specific T cell function and phenotype.

Purpose of the Study:

• To discuss the application and experience with a standard protocol for intracellular cytokine staining.
• To highlight the importance of this technique in evaluating vaccine efficacy and infectious disease research.

Main Methods:

• Flow cytometry technique involving short-term culture of stimulated immune cells.
• Use of protein secretion inhibitors, followed by fixation and permeabilization.
• Staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies.

Main Results:

• This method is highly popular for assessing antigenicity due to its ability to provide simultaneous functional and phenotypic data.
• Modern flow cytometers allow for the detection of numerous colors, enhancing the complexity of analyses.

Conclusions:

• Intracellular cytokine staining is an indispensable tool in immunology, particularly for vaccine development and infectious disease research.
• The described standard protocol facilitates robust measurement of antigen-specific immune responses.