Application Note Cell Analysis Analysis of Phosphorylated STAT Protein Signaling in Lymphocytes Using Flow Cytometry Author Abstract Agilent Technologies, Inc. This application note examines the phosphorylation of signal transducer and activator of transcription (STAT) proteins in response to cytokine stimulation using a flow cytometry–based phosphoflow technique and the Agilent NovoCyte flow cytometer. The results demonstrate the efficacy of this approach for the rapid, quantitative, scalable, and multiparameter detection of phosphorylated protein in single cells. Introduction many cellular events including T and blotting, and mass spectrometry. B cell signaling, cell metabolism, cell Western blotting is the most commonly Protein phosphorylation is the growth, apoptosis, and other processes. used but has shortcomings; it is biological process of transferring semiquantitative, time-consuming, and Cytokines are a group of small a phosphate group to a substrate requires a large amount of starting secreted proteins important for protein, which primarily occurs on material. Also, cell separation may be immune cell-to-cell communication, tyrosine, serine, and threonine residues. required to isolate a pure population of a immune cell activation, differentiation, Protein phosphorylation can cause cells from a heterogeneous mixture. conformational changes, changes and migration towards the site of Analysis of phosphorylated proteins by in protein activity, or protein-protein inflammation/infection. Cytokines bind to flow cytometry or phosphoflow was interactions. This event can also initiate receptors on the cell surface and activate first described in the early 2000s. By a phosphorylation signaling cascade intracellular cascades, such as the Janus combining a phosphoflow methodology leading to a sequence of protein kinase (JAK)/STAT pathway. STATs are with cell surface antibody staining, rare phosphorylation events. the final phosphorylated protein in this cascade; they translocate to the nucleus cell populations can be studied in the This produces an assortment of kinases and alter gene expression. There are presence of other cell types. Multiple and phosphatases, which regulate many methods for detecting intracellular cell types can also be analyzed in intracellular protein phosphorylation. protein phosphorylation, including one sample. Protein phosphorylation is crucial for radiometric kinases assays, western 1.1 105 105.5 A 309.2 1.0 240 104 ) ) 3 6 0.8 4 0 0 10 180 0.6 120 CD16 PE-H FSC-A (1 SSC-H (1 CD19 APC-H 3 10 3 0.4 10 60 0 0 0 0.2 -103 -103 0.1 0.4 0.8 1.2 1.6 2.0 2.3 0.1 0.4 0.8 1.2 1.6 2.0 2.3 -102 103 104 105 -102 103 104 105 FSC-H (106) FSC-H (106) CD3 Pacific Blue-H CD3 Pacific Blue-H B T cells CD3+ B cells CD19+ NK cells CD16+ C Medians fold-change 25 T cells B cells 0 12.525 NK cells 20 unstim 1 min. 15 2 min. 5(pY694) 4 min. AT 10 ST 8 min. 12 min. 5 16 min. -103 103 104 105 -103 103 104 105 -103 103 104 105 p-stat5 Ax647-H p-stat5 Ax647-H p-stat5 Ax647-H 0 0 10 20 Time of stimulation (min) Figure 1. Time-dependent differences in IL-2–induced STAT5 phosphorylation among lymphocyte subsets. PBMCs were isolated from the blood of a healthy donor. Cells were stained with αCD19 FITC antibody before IL-2 stimulation. PBMCs were then stimulated with 100 ng/mL IL-2 for 0 to 16 minutes at 37 °C. After cytokine stimulation, cells were fixed in 1.5% paraformaldehyde for 10 minutes at room temperature and pelleted by centrifugation. Thereafter, cells were permeabilized with ice cold 100% methanol for 15 minutes. Finally, cells were stained with αCD16 PE, αCD3 Pacific Blue, and α-p-STAT5 AlexaFluor647 for one hour. The main cell population was identified by FSC-H and SSH-H. Single cells were than identified in the FSC-H and FSC-A plot to eliminate doublets. Cell surface stains identified the lymphocyte populations: CD19 B cells, CD3 T cells, and CD16 NK Cells. Data were analyzed using the CytoBank website for histogram overlay and heat map analysis (B). Changes in p-STAT5 expression were graphed as the fold change/time compared to the unstimulated sample for each lymphocyte population (C). 2 IL-2 induces potent cellular structure. This was followed by any indications of response, even when measured at the longest time point. Both p-STAT5 signaling in T and permeabilization with 100% methanol to allow the phospho-specific antibodies T cells and NK cells responded rapidly, NK but not B lymphocyte to stain inside the cell. Surface stains showing a 4.5- and 3.2-fold increase, cell populations identified specific population of respectively, in p-STAT5 levels within one lymphocytes: CD19 for B cells, CD3 minute of IL-2 addition. The phosphorylation of STAT5 in for T cells, and CD16 for Natural Killer Although time-dependent increases were peripheral blood mononuclear cells (NK) cells as shown in the sample sustained in T cells, no further increases (PBMCs) when induced by IL-2 was gating plots (Figure 1). Since the CD19 in p-STAT5 were seen in NK cells investigated to examine B, T, and NK epitope was destroyed upon exposure to after eight minutes. This result can be lymphocyte cell populations. The degree formaldehyde, the PBMCs were stained explained by the differential expression of phosphorylation was determined by with αCD19 antibody before cytokine of IL-2 receptors in lymphocyte phosphoflow, in which a phosphorylated stimulation. populations, as B cells express relatively protein-specific antibody is used to PBMCs were treated with 100 ng/mL few IL-2 receptors compared to T and determine the levels of p-STAT5 (Y694). of IL-2 from 0 to 16 minutes, stained NK cells. IL-2 receptor expression PBMCs were either stimulated with IL-2 for p-STAT5, and then analyzed on the is especially high in activated T cell or IL-4 for a varying amount of time or NovoCyte flow cytometer (Figure 1). populations that are particularly reliant concentration of cytokine. Following a p-STAT5 levels varied greatly in T, B, and on IL-2 signaling for cell survival and typical fixation/permeabilization protocol NK cell populations. Both T and NK cells proliferation. for phosphoflow, cells were first fixed in responded, while B cells did not show 1.5% paraformaldehyde to stabilize the A T cells C3 B cells C19 NK cells C16 B 30 edians fold-change T cells 0 12.5 25 25 N cells 20 unstim 0.006 ngm 15 0.032 ngm 0.16 ngm 10 0.8 ngm 4 ngm median fold-change STAT5p694 5 20 ngm 0 100 ngm 0 -2 -1 0 1 2 3 4 og10I-2 ngm 500 ngm -103 103 104 105 -103 103 104 105 -103 103 104 105 p-STAT5 A64- p-STAT5 A64- p-STAT5 A64- Figure 2. Dose-response analyses reveal differences in IL-2–induced STAT5 phosphorylation among lymphocyte subsets. PBMCs were isolated and stained with αCD19 FITC, as described in Figure 2. Cells were stimulated with IL-2 at a concentration of 0 to 500 ng/mL for 15 minutes. PBMCs were then fixed and permeabilized, as described in Figure 2. Data was analyzed using the tools provided by the CytoBank website for histogram overlay and heat map analysis of p-STAT5 phosphorylation results (A). Changes in p-STAT5 expression were then recorded as fold change compared to unstimulated sample, with increasing concentration of IL-2 for the T and NK cell population (B). EC50 values of STAT5 phosphorylation of T and NK cells were determined by Prism 6 software. B cells were not graphed as they did not respond to IL-2 stimulation. 3 Next, the phosphorylation of STAT5's IL-4 induces robust p-STAT6 expression at the Y641 residue dose-dependent response to IL-2 in in T, B, and NK cell populations (Figure 3). PBMCs was investigated. Cells were p-STAT6 signaling in All lymphocyte populations responded to treated with 0 to 500 ng/mL of IL-2 all lymphocyte cell IL-4, but B cells were the most sensitive, for 15 minutes followed by p-STAT5 displaying a significant increase in analysis in each lymphocyte subset populations p-STAT6 with as little as 0.16 ng/mL of (Figure 2). A stronger dose-dependent IL-4 is another important cytokine in IL-4. No significant increase was seen in response for T cells was observed than the immune response vital for T cell T and NK cells until IL-4 concentration that of NK cells. While NK cells did not differentiation, immunoglobulin class reached 4 ng/mL. respond to increases in IL-2 after a switching in B cells, and alternative The p-STAT6 response in B cells also concentration of 20 ng/mL, increases in macrophage activation. IL-4 is primarily became saturated at a much lower p-STAT5 levels in T cells were seen at the secreted by activated T cells, basophils, concentration of IL-4 (20 ng/mL), while highest concentration tested, 500 ng/mL; and mast cells. IL-4 signaling results in in T and NK cells, the saturation of the calculated EC50 values of STAT5 a rapid increase in phosphorylation of p-STAT6 abundance was observed up phosphorylation of IL-2 stimulated T cells STAT6 and plays an important role in to 100 ng/mL. This was underscored and NK cells were 21.57 ng/mL and 3.89 the adaptive immune response of helper by the EC50 values calculated for ng/mL, respectively (Figure 2B). The T cells and B cells. the lymphocyte populations: B cells observed by p-STAT5 analysis by flow 2.58 ng/mL, T cells 17.51 ng/mL, and cytometry showed that T cells respond In this next experiment, we examined the NK cells 19.39 ng/mL (Figure 3B).