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Laurie Lamoreaux¹, Mario Roederer, Richard Koup

¹Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, USA. llamorea@mail.nih.gov

Nature Protocols

|April 5, 2007

Summary

This study optimizes polychromatic flow cytometry for analyzing human T lymphocytes. The method enhances simultaneous detection of multiple cell functions and surface markers, improving immunophenotyping accuracy.

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Area of Science:

Immunology
Cell Biology
Biotechnology

Background:

Polychromatic flow cytometry is a powerful tool for analyzing immune cells.
Optimizing multicolor panels for human T lymphocyte surface and intracellular staining is complex.
Standard protocols may not capture the full functional capacity of T cells.

Purpose of the Study:

To present a method for optimizing polychromatic flow cytometry (up to 17 fluorochromes) for human T lymphocyte analysis.
To detail procedural optimization of key steps for intracellular cytokine staining assays.
To establish a polychromatic assay for detecting five T lymphocyte functions and multiple surface markers.

Main Methods:

Systematic optimization of monoclonal antibody titration.
Inclusion of dead-cell discriminators and 'dump' channels.
Careful selection of cytokine secretion inhibitors, fixation/permeabilization reagents, and compensation controls.
Development of a multiplex assay for simultaneous functional and phenotypic analysis.

Main Results:

A refined protocol for polychromatic flow cytometry of human T lymphocytes.
Successful simultaneous detection of three cytokine productions, one chemokine production, and degranulation.
Simultaneous identification of multiple T lymphocyte surface markers.

Conclusions:

The described method provides a robust framework for optimizing polychromatic flow cytometry assays.
This approach significantly enhances the ability to dissect complex T lymphocyte responses.
The established assay allows for comprehensive immunophenotyping and functional profiling of T cells.