Intracellular Flow

With the ability to collect data at the level of individual cells, flow cytometry offers several advantages for cell signaling studies.

Unlike lysate-based approaches, flow cytometry facilitates the detection and analysis of heterogeneous signaling responses. Thus, it is possible to distinguish between a robust protein phosphorylation response within a small population of cells versus a smaller but more homogeneous response. With the addition of fluorescent antibodies specific for cell surface markers to cell subsets within complex cell mixtures such as whole blood, signaling responses mediated by protein phosphorylation can be detected. Moreover, rare cell populations can be uncovered without pre-enrichment of the cells. As a result, rich data is obtained from limited cell samples.


BD Phosflow™ products consist of a system of buffers and fluorescent monoclonal antibodies optimized for the flow cytometric detection of intracellular signaling molecules and specific post-translational modifications. To detect specific phosphorylated epitopes by flow cytometry, cells are fixed to maintain the phosphorylated state of signaling proteins and then permeabilized to allow antibodies to enter cells and specifically bind to target proteins. Flow cytometric analysis of stained cells can then capture a “snapshot” of intracellular signaling and protein phosphorylation.

Protein phosphorylation is transient by nature and heavily regulated by protein phosphatases. Appropriate stimulation time points and prompt inactivation of phosphatases are required for most methods of phosphoprotein detection, including Western blot analysis. Likewise, cell samples for flow cytometric analysis must be quickly fixed to maintain phosphoepitopes. Another important consideration is the level of phosphoprotein expression. Certain protein phosphorylation events can be difficult to detect at the single-cell level, particularly when cells express low levels of a particular signaling protein or exhibit incomplete phosphorylation of the site of interest. BD offers high-quality antibodies directly conjugated to bright fluorochromes to greatly improve the intracellular detection of phosphoprotein.


Enhanced IL-2 sensitivity of Tregs

Enhanced IL-2 sensitivity of Tregs

Human PBMCs were stained with Alexa Fluor® 647 Anti-Human CD127 antibody during a 15-minute stimulation with 0-, 0.01-, 0.1-, 1-, 10-, or 100-ng/mL doses of recombinant human IL-2. Cells were fixed using BD Cytofix fixation buffer, permeabilized using BD Phosflow perm buffer III, and stained with fluorescent antibodies specific for Stat5 (pY694), CD4, CD8, and CD25. Samples were acquired using a BD LSRFortessa™ flow cytometry system and analyzed using Cytobank software. CD4 T-cell subsets were identified as shown. Compared to other T cells, Tregs phosphorylate Stat5 in response to much lower concentrations of IL-2.

Enhanced IL-2 sensitivity of Tregs.

Human PBMCs were stained with Alexa Fluor® 647 Anti-Human CD127 antibody during a 15-minute stimulation with 0-, 0.01-, 0.1-, 1-, 10-, or 100-ng/mL doses of recombinant human IL-2. Cells were fixed using BD Cytofix fixation buffer, permeabilized using BD Phosflow perm buffer III, and stained with fluorescent antibodies specific for Stat5 (pY694), CD4, CD8, and CD25. Samples were acquired using a BD LSRFortessa™ flow cytometry system and analyzed using Cytobank software. CD4 T-cell subsets were identified as shown. Compared to other T cells, Tregs phosphorylate Stat5 in response to much lower concentrations of IL-2.


Multiparameter flow cytometry offers key advantages for the detection and analysis of intracellular signaling within cells and cell subsets in complex mixtures. Since cell signaling studies often examine cellular responses to treatment with stimulatory or inhibitory molecules, unstimulated or untreated cells often provide the best controls for evaluating background staining. Unlike an immunoglobulin isotype control, an unstimulated cell control takes into account the unique background characteristics of each antibody as well as the basal phosphoprotein expression levels within the cells of interest. Cellular permeabilization is required to expose phosphorylated epitopes. Although multiple buffer systems are available, BD Phosflow perm buffer III is recommended for most applications. The BD Biosciences website contains useful information about the performance of various BD cell surface marker and intracellular antibodies in different buffer conditions and staining protocols.

BD Phosflow kits provide useful tools for analyzing the signaling responses elicited by cells of the adaptive and innate immune systems. These kits include fluorescent antibody cocktails specific for cell surface markers to identify specific leucocyte subsets as well as fluorescent antibodies to key phosphoproteins. In addition, the kits provide compatible buffers, positive and negative control cells, and detailed protocols. The BD Phosflow™ human monocyte/NK cell activation kit (Cat. No. 562089) allows the simultaneous study of protein phosphorylation in B cells, T cells, monocytes, and NK cells from human whole blood, while the BD Phosflow™ human T-cell activation kit (Cat. No. 560750) is useful for the study of phosphoprotein responses in CD4 versus CD8 T cells.


Stat5 and Stat6 signaling responses to IL-4 differ among human leucocyte subsets

Stat5 and Stat6 signaling responses to IL-4 differ among human leucocyte subsets

Human whole blood was stimulated with human IL-4 and fixed, permeabilized, and stained using the BD Phosflow human monocyte/NK cell activation kit. Samples were acquired and analyzed by flow cytometry using a BD LSRFortessa™ cell analyzer, and then analyzed using Cytobank software. Leucocyte subsets were identified as shown. Analysis of Stat6 (pY641) and Stat5 (pY694) phosphorylation responses to IL-4 revealed that all four leucocyte subsets responded to IL-4 by phosphorylating Stat6. In contrast, Stat5 responses varied considerably among cell types, with NK cells not showing a detectable Stat5 phosphorylation response.

Stat5 and Stat6 signaling responses to IL-4 differ among human leucocyte subsets

Human whole blood was stimulated with human IL-4 and fixed, permeabilized, and stained using the BD Phosflow human monocyte/NK cell activation kit. Samples were acquired and analyzed by flow cytometry using a BD LSRFortessa™ cell analyzer, and then analyzed using Cytobank software. Leucocyte subsets were identified as shown. Analysis of Stat6 (pY641) and Stat5 (pY694) phosphorylation responses to IL-4 revealed that all four leucocyte subsets responded to IL-4 by phosphorylating Stat6. In contrast, Stat5 responses varied considerably among cell types, with NK cells not showing a detectable Stat5 phosphorylation response.


1 Szabo SJ, Sullivan BM, Stemmann C, Satoskar AR, Sleckman BP, Glimcher LH. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science. 2002;295:338-342.

2 Intlekofer AM, Takemoto N, Wherry EJ, et al. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol. 2005;6:1236-1244.