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Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET
Published on: January 27, 2012
Protocol for measuring cell cycle Zn2+ dynamics using a FRET-based biosensor
Samuel E Holtzen1, Ananya Rakshit2, Amy E Palmer2
1Molecular, Cellular & Developmental Biology, University of Colorado Boulder, Boulder, CO 80309, USA; Department of Biochemistry and BioFrontiers Institute, 3415 Colorado Avenue, University of Colorado Boulder, Boulder, CO 80303, USA.
STAR Protocols
|June 11, 2024
Summary
This study details a method for long-term live-cell imaging to track zinc (Zn2+) fluctuations within cells. The protocol allows researchers to observe how cellular zinc levels change naturally and in response to external factors.
Area of Science:
- Cellular Biology
- Biochemistry
- Microscopy
Background:
- Cellular zinc (Zn2+) levels are dynamic, not static.
- Zn2+ pools fluctuate naturally during the cell cycle and in response to stimuli.
Purpose of the Study:
- To present a protocol for long-term live-cell imaging of cytosolic Zn2+.
- To enable analysis of single-cell Zn2+ traces in response to perturbations.
Main Methods:
- Utilizing live-cell imaging with a cytosolic Zn2+ sensor.
- Employing the EllipTrack pipeline for cell tracking.
- Analyzing single-cell traces to quantify labile Zn2+ changes.
Main Results:
- Successfully implemented a protocol for long-term live-cell imaging of Zn2+ dynamics.
- Developed a method to track and analyze cellular Zn2+ responses to perturbations.
Conclusions:
- The presented protocol facilitates detailed investigation of cellular Zn2+ homeostasis.
- This method allows for the study of dynamic Zn2+ changes in single cells over time.
