Intracellular Phospho-protein Staining Techniques for Flow Cytometry: Monitoring Single Cell Signaling Events

Department of Microbiology and Immunology, Stanford University, Stanford, California, United States
Cytometry Part A (Impact Factor: 2.93). 10/2003; 55(2):61-70. DOI: 10.1002/cyto.a.10072
Source: PubMed


Recent advances in intracellular staining techniques, cytometer technology, fluorescent reagents, and antibody production have expanded the number of intracellular antigens that can be analyzed by flow cytometry. Measurement of protein phosphorylation with phospho-specific antibodies has given insight into kinase signaling cascades. However, available techniques for phospho-epitope staining can differ greatly, making it necessary to understand the differences between the outcomes when such techniques are applied and to develop robust and reproducible methods of application.
Ten different cellular fixation and permeabilization techniques were tested for their ability to provide phospho-specific staining. Combinations of formaldehyde, methanol, ethanol, acetone, Triton X-100, and saponin were used as fixation and permeabilization reagents. Phospho-specific antibodies were labeled with Alexa Fluor dyes to provide multicolor analysis of different signaling events simultaneously within individual cells.
Fixing cells with 1.5% formaldehyde followed by permeabilization in methanol gave optimal results for pERK, pp38, pJNK, pStat1, pStat5, and pStat6 staining. Alteration of formaldehyde fixation and methanol permeabilization times affected measurements of phosphorylation induction. Phospho-specific flow cytometric analyses correlated well with Western blotting, providing cross platform validation of the technique.
Measuring phosphorylation events by flow cytometry provides a rapid and efficient way to measure kinase cascades in individual cells. Stability of phospho-epitopes in methanol allows long-term storage of samples prior to analysis. Multiple signaling cascades can be monitored simultaneously through the use of different fluorophore labels to determine specificity of ligands or inhibitors. Application of optimized techniques to heterogeneous cell types such as peripheral blood or murine splenocytes may allow signaling to be analyzed simultaneously in immune cell subsets.

  • Source
    • "One practical application of this gating strategy is the study of the signaling pathways present in these 5 subsets using intracellular phosphoflow staining (Krutzik and Nolan, 2003). We optimized isolation conditions and antibodies for use with the necessary PFA fixation and methanol permeabilization. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune- and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR-ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3 DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.
    Full-text · Article · Sep 2015 · Journal of immunological methods
  • Source
    • "Primity Bio Pathway Phenotyping UT-7-EpoR cells were starved overnight; stimulated with saturated concentrations of EPO and the indicated diabodies for 15, 60, and 120 min; and fixed with 1% PFA for 10 min at room temperature. The fixed cells were prepared for antibody staining according to standard protocols (Krutzik and Nolan, 2003). Briefly, the fixed cells were permeabilized in 90% methanol for 15 min. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to “tune” signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.
    Full-text · Article · Mar 2015 · Cell
  • Source
    • "A DMSO concentration of 0.1% used in assays is reported to have less influence in hES cell differentiation than higher concentrations of DMSO [26] [27]. After harvesting with 0.05% trypsin-EDTA solution (PAA Laboratories, Linz, Austria) and washing with PBS, single hES cell suspensions were fixed using 1.6% paraformaldehyde (PFA, Sigma-Aldrich) for 10 min at room temperature (RT) as described for the detection of intracellular phosphoproteins [28] [29]. Cells were then washed with permeabilisation buffer (Foxp3 Staining Buffer Set, e-Biosciences). "
    [Show abstract] [Hide abstract]
    ABSTRACT: As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation.When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.
    Full-text · Article · Nov 2014 · International Journal of Cell Biology
Show more