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A simple method for incorporating aequorin into mammalian cells
The American Journal of Physiology
|August 1, 1986
Summary
A new, simple method effectively incorporates aequorin into mammalian cells for measuring cytosolic ionized calcium (Ca2+). This technique offers a less invasive alternative to existing methods like scrape loading and hypoosmotic treatment (HOST).
Area of Science:
- Cell biology
- Biochemistry
- Physiology
Background:
- Accurate measurement of cytosolic ionized calcium (Ca2+) is crucial for understanding cellular signaling.
- Existing methods for introducing calcium indicators like aequorin into cells have limitations.
Purpose of the Study:
- To develop and validate a simple, efficient method for incorporating aequorin into mammalian cells.
- To compare the efficacy of the new method with established techniques.
Main Methods:
- A novel procedure involving cell incubation with aequorin, followed by centrifugation.
- Comparison of aequorin incorporation levels with scrape loading and hypoosmotic treatment (HOST).
Main Results:
- The new method achieves similar aequorin incorporation to scrape loading but significantly reduces it compared to HOST (by 70%).
- Successful application of the method in various cell types including hepatocytes, kidney cells, macrophages, and cardiac myocytes.
Conclusions:
- This simple centrifugation-based method provides an effective means to load mammalian cells with aequorin for cytosolic Ca2+ measurement.
- The technique is less disruptive than HOST, making it suitable for a range of cell types.