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Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
Published on: January 6, 2016
Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events
Peter O Krutzik1, Garry P Nolan
1Department of Molecular Pharmacology, School of Medicine, Stanford University, Stanford, California 94305, USA.
Insights
Optimizing fixation and permeabilization for phospho-specific flow cytometry enhances kinase cascade analysis in single cells. This robust method allows for simultaneous monitoring of multiple signaling pathways and long-term sample storage.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Intracellular staining, flow cytometry, and reagent advancements enable broader intracellular antigen analysis.
- Phospho-specific antibodies offer insights into kinase signaling pathways.
- Variability in phospho-epitope staining techniques necessitates robust and reproducible methods.
Purpose of the Study:
- To evaluate and optimize cellular fixation and permeabilization techniques for phospho-specific flow cytometry.
- To establish reproducible methods for analyzing intracellular phosphorylation events.
Main Methods:
- Tested ten different fixation (formaldehyde, methanol, ethanol, acetone) and permeabilization (Triton X-100, saponin) techniques.
- Utilized phospho-specific antibodies labeled with Alexa Fluor dyes for multicolor analysis.
- Compared flow cytometry results with Western blotting for validation.
Main Results:
- Optimal phospho-specific staining for pERK, pp38, pJNK, pStat1, pStat5, and pStat6 achieved with 1.5% formaldehyde fixation and methanol permeabilization.
- Fixation and permeabilization times influenced phosphorylation induction measurements.
- Flow cytometry results showed strong correlation with Western blotting.
Conclusions:
- Flow cytometry offers a rapid and efficient method for measuring kinase cascades in single cells.
- Methanol-based permeabilization allows for stable phospho-epitope storage.
- Simultaneous monitoring of multiple signaling pathways is feasible using different fluorophores.
- Optimized techniques can be applied to heterogeneous cell types for immune cell subset analysis.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
