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. 2017 Aug 4;73:18.19.1-18.19.14.
doi: 10.1002/cptx.26.

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells

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Free PMC article

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells

Fredine T Lauer et al. Curr Protoc Toxicol. .
Free PMC article

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.

Keywords: HPBMC; flow cytometry; immunophenotyping; intracellular staining; toxicology.

Figures

Figure 1
Figure 1
Four major Th subsets and cytokines and transcription factors produced by them.
Figure 2
Figure 2
Schematic of the major steps in this procedure.
Figure 3
Figure 3
Depiction of the gating strategy and the specific antigen used to identify each subset of cells.
Figure 4
Figure 4
Examples of gating strategy using dot plots created in FlowJo V10 software.
Figure 5
Figure 5
Normal HPBMC in stimulated and unstimulated conditions. Black bars represent freshly isolated and analyzed HPBMC, whereas red and black bars represent HPBMC analyzed following short term cryopreservation (Cryo stored 30–90 days) and long term cryopreservation (>180 days). Data revealed significant differences in the CD3/CD69 phenotype of the short term cryopreserved in both the stimulated and unstimulated samples (p values = 0.012 and 0.010 respectively) (A,C). No significant differences were seen in unstimulated HPBMC between fresh and cryopreserved samples (C,D). Groups were N=7 for fresh, n=6 for short term cryopreserved, and N=4 for long term cryopreserved. Data shown as mean +/- standard deviation. Analysis of HPBMC was performed using multicolor flow cytometry (FlowJo software). Statistical analysis (One Way ANOVA followed by Dunnett’s Method for multiple comparisons versus control group) performed using SigmaPlot version 12.5.

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