Related Experiment Videos
Detecting mitochondrial permeability transition by confocal imaging of intact cells pinocytically loaded with calcein
Rachel A Jones1, Alison Smail, Mark R Wilson
1Department of Biological Sciences, University of Wollongong, New South Wales, Australia.
Abstract:
When studied in vitro, mitochondrial permeability transition (MPT) is associated with an increase in mitochondrial permeability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane (Deltapsimit). The MPT has been implicated as being important in cellular calcium homeostasis, autophagy and cell death via necrosis and apoptosis. Thus, it is important to develop a valid technique for accurate measurement of this phenomenon in intact cells. We developed a procedure for the detection of MPT in intact cells that avoids the disadvantages associated with earlier approaches. In this new technique, unmodified (green-fluorescent) calcein is simultaneously introduced into the cytosol of millions of cells by the process of pinocytic loading and, to identify the position of individual mitochondria and to measure Deltapsimit, the cells are counter-stained with a red-fluorescing potentiometric dye. Using this approach with a variety of cell types, we demonstrate that cytosolic calcein is excluded from normal polarized mitochondria but enters them during MPT. This technique may be valuable in studies investigating the cellular functions of MPT.
Insights
Researchers developed a new method to measure mitochondrial permeability transition (MPT) in live cells. This technique accurately detects MPT, a key process in cell death and calcium regulation.
Area of Science:
- Cell Biology
- Mitochondrial Physiology
- Biochemistry
Background:
- Mitochondrial permeability transition (MPT) involves increased mitochondrial permeability and loss of inner membrane potential (Δψmit).
- MPT is crucial for cellular calcium homeostasis, autophagy, and programmed cell death (apoptosis and necrosis).
- Accurate measurement of MPT in intact cells is essential for understanding its cellular roles.
Purpose of the Study:
- To develop a novel, valid technique for detecting MPT in intact cells.
- To overcome limitations of previous MPT measurement methods.
- To facilitate studies on the cellular functions of MPT.
Main Methods:
- Simultaneous pinocytic loading of green-fluorescent calcein into the cytosol of millions of cells.
- Counter-staining with a red-fluorescing potentiometric dye to visualize mitochondria and measure Δψmit.
- Utilizing calcein exclusion/entry into mitochondria as an indicator of MPT.
Main Results:
- The developed technique successfully detects MPT in various cell types.
- Normal polarized mitochondria exclude cytosolic calcein.
- Calcein enters mitochondria during MPT, indicating increased permeability.
Conclusions:
- A new, reliable method for measuring MPT in intact cells has been established.
- This technique offers advantages over existing approaches.
- The method is valuable for investigating MPT's role in cellular processes.