Intracellular Flow

While other techniques can measure either cytokines, transcription factors, or phosphorylated proteins separately, intracellular flow cytometry enables the measurement of multiple intracellular markers simultaneously at the singlecell level.

This method provides data on signaling responses, differentiation states, and other cellular events. The combined use of fluorescent antibodies specific for cell surface and intracellular markers enables high resolution comparative analyses of the phenotypic and functional differences within multiple cell types across samples.


In the example shown, cells from human whole blood were analyzed for Stat5 phosphorylation induced by IL-2 stimulation. Different populations of naïve, effector, and memory T cells were identified using phenotypic markers. In addition to cell surface markers, phenotyping was aided by analysis of T-bet, a signature transcription factor involved in interferon-γ production and known to be expressed in NK cells, CD8 effector T cells, and CD4 T helper 1 (Th1) cells.1,2

In this experiment, differences in IL-2 sensitivity were observed across the different cell types. For instance, Th1-like and non-Th1 effector/memory CD4 T-cell subsets (populations F and E, respectively) responded to stimulation with very low concentrations of IL-2, whereas naïve CD4 T cells (population D) and naïve CD8 T cells (population A) required higher doses of IL-2 to induce Stat5 phosphorylation.


IL-2 dose-response in T-cell subsets

IL-2 dose-response in T-cell subsets

Human whole blood was stimulated with various concentrations of human recombinant IL-2 (0.05–100 ng/mL) for 15 minutes. Cells were fixed with BD Phosflow™ lyse/fix buffer, permeabilized with BD Phosflow perm buffer III, and stained with fluorescent antibodies specific for CD3, CD4, CD45RA, T-bet, and Stat5 (pY694). Samples were analyzed using a BD LSR II flow cytometry system with Cytobank software. T-cell subsets were identified as shown.

IL-2 dose-response in T-cell subsets

Human whole blood was stimulated with various concentrations of human recombinant IL-2 (0.05–100 ng/mL) for 15 minutes. Cells were fixed with BD Phosflow™ lyse/fix buffer, permeabilized with BD Phosflow perm buffer III, and stained with fluorescent antibodies specific for CD3, CD4, CD45RA, T-bet, and Stat5 (pY694). Samples were analyzed using a BD LSR II flow cytometry system with Cytobank software. T-cell subsets were identified as shown.


The simultaneous measurement of the expressed levels of multiple transcription factors, cytokines, and surface markers by cells within the same sample is very useful for the study of T helper (Th) cell differentiation and function. RORγt is the signature transcription factor for Th17 cells. It is important for the secretion of IL-17 and the maintenance of CD4+CD8+ double-positive thymocytes.1,2

In the experimental results shown, cells were isolated from BALB/c mouse thymus and spleen. Thymocytes or splenocytes were surface stained with fluorescent antibodies specific for CD44, CD62L, CD196, or appropriate immunoglobulin isotype controls. Cells were fixed and permeabilized with the BD Pharmingen transcription factor buffer set and then intracellularly stained with fluorescent antibodies specific for RORγt, Foxp3, IL-17A, and IFN-γ.

In the top panel, the cellular expression of IL-17A is compared with the expression of RORγt, Foxp3 (Treg transcription factor) and IFN-γ (Th1 cytokine). As expected in thymocytes, there were many IL-17A+ RORγt+ double-positive cells, while there was essentially no co-expression of IL-17A with Foxp3 or IFN-γ. Splenocytes expressed very little IL-17A.

In the bottom panel (B), further analysis revealed that IL-17A– producing cells from spleen expressed the surface markers CD44 and CD196 (CCR6) but not CD62L, providing additional information on the phenotype of IL-17A+ cells.


Phenotypic analysis of Th17 cells from BALB/c thymus and spleen

Phenotypic analysis of Th17 cells from BALB/c thymus and spleen

Thymocytes or splenocytes were surface stained with fluorescent antibodies specific for CD44, CD62L, CD196, or appropriate immunoglobulin isotype controls. Cells were fixed and permeabilized with the BD Pharmingen transcription factor buffer set, then intracellularly stained for RORγt, Foxp3, IL-17A, and IFN-γ.

Phenotypic analysis of Th17 cells from BALB/c thymus and spleen

Thymocytes or splenocytes were surface stained with fluorescent antibodies specific for CD44, CD62L, CD196, or appropriate immunoglobulin isotype controls. Cells were fixed and permeabilized with the BD Pharmingen transcription factor buffer set, then intracellularly stained for RORγt, Foxp3, IL-17A, and IFN-γ.


1 Ghoreschi K, Laurence A, Yang XP, Hirahara K, O’Shea JJ. T helper 17 cell heterogeneity and pathogenicity in autoimmune disease. Trends Immunol. 2011;32:395-401.

2 Ivanov II, McKenzie BS, Zhou L, et al. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006;126:1121-1133.