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Updated: Feb 25, 2026
Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis
Published on: October 17, 2018
Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T
Fredine T Lauer1, Jesse L Denson1, Ellen Beswick2
1Department of Pharmaceutical Sciences, College of Pharmacy, The University of New Mexico, Albuquerque, New Mexico.
Insights
This study details methods for detecting intracellular markers in activated CD4+ T-cells using flow cytometry. Cryopreservation of human peripheral blood mononuclear cells (HPBMC) generally preserves cell phenotypes, with minor exceptions.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Previous work established protocols for human peripheral blood mononuclear cells (HPBMC) isolation and cryopreservation.
- Accurate immunophenotyping of HPBMC is crucial for understanding immune responses.
Purpose of the Study:
- To describe a multi-color flow cytometry method for detecting intracellular markers (ICM) in activated CD4+ T-cells.
- To evaluate the impact of cryopreservation on HPBMC phenotypes, including intracellular markers.
Main Methods:
- Activation and differentiation of CD4+ T-cells.
- Multi-color flow cytometry for cell surface marker (CSM) and intracellular marker (ICM) detection.
- Analysis of eight specific HPBMC phenotypes in fresh versus cryopreserved samples.
Main Results:
- Repeatable and robust detection of ICM in surface marker-defined T-cells was achieved.
- No significant differences were observed in eight analyzed HPBMC phenotypes between fresh and cryopreserved samples (up to 90 days).
- A minor difference was noted in activated T- CD3/CD69 expression in short-term cryopreserved samples.
Conclusions:
- The described flow cytometry protocol enables reliable detection of ICM in functional T-cell subsets.
- Cryopreservation is a viable method for storing HPBMC for immunological studies, with minimal impact on most analyzed phenotypes.
Abstract:
In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi-color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T-cells using multi-color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker-defined T cells that identify functional subsets of cells. There were no observed differences between fresh and cryopreserved HPBMC in eight phenotypes analyzed (T-CD3, Th-CD4, Tmem-CD45RO, activated T-CD3/CD25, Treg- Foxp3/CD25, Th1-IFNγ, Th2- IL-4, Th17-IL-17A). There was an observed difference in activated T- CD3/CD69 in the short term (30-90 days) cryopreserved samples as compared to the freshly isolated samples, which may have resulted from the variance in controls or small sample size. © 2017 by John Wiley & Sons, Inc.
