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Detection of kinase translocation using microfluidic electroporative flow cytometry.
Jun Wang1, Ning Bao, Leela L Paris
1Departments of Agricultural and Biological Engineering, School of Chemical Engineering, Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907, USA.
Analytical Chemistry
|December 25, 2007
Summary
A new method, microfluidic electroporative flow cytometry, detects kinase translocation in B cells. This technique differentiates stimulated cells by measuring kinase release, aiding drug discovery and diagnostics.
Area of Science:
- Cell Biology
- Biotechnology
- Immunology
Background:
- Kinase localization is crucial for cell signaling and activation.
- Conventional methods for detecting kinase translocation lack high throughput and single-cell resolution.
- Subcellular fractionation and Western blotting provide population averages, not individual cell behavior.
Purpose of the Study:
- To develop a novel high-throughput method for detecting kinase translocation in single cells.
- To assess the utility of microfluidic electroporative flow cytometry for analyzing protein localization.
- To investigate the translocation of tyrosine kinase Syk in B cells.
Main Methods:
- Development of microfluidic electroporative flow cytometry.
- Combining electroporation with flow cytometry to measure intracellular kinase release.
- Utilizing an EGFP-tagged tyrosine kinase (Syk) in B cells.
Main Results:
- Kinase release during electroporation is influenced by subcellular localization.
- Stimulated B cells with Syk at the plasma membrane retained more kinase.
- The technique successfully differentiated cell populations based on kinase translocation.
- Single-cell analysis of kinase translocation within a population was achieved.
Conclusions:
- Microfluidic electroporative flow cytometry enables high-throughput, single-cell analysis of protein translocation.
- This method can distinguish between stimulated and unstimulated cell populations.
- The technique holds potential for kinase-related drug discovery, cancer diagnosis, and staging.
