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

Department of Molecular Pharmacology, School of Medicine, Stanford University, Stanford, California 94305, USA.
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.

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    • "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. "
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    • "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. "
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    • "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). "
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