Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of …
CBL Campos, BA Paim, RG Cosso… - Cytometry Part A …, 2006 - Wiley Online Library
Cytometry Part A: The Journal of the International Society for …, 2006•Wiley Online Library
Background: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis
and is used to characterize the mitochondria‐dependent pathway of this type of cell death.
Techniques currently used to measure cytochrome c release, Western blot and fluorescence
microscopy of immunolabeled cells, are time‐consuming and inaccurate, and the latter is
still limited by sample size. Methods: We developed a rapid and reliable technique to detect
cytochrome c release during drug‐induced apoptosis, using flow cytometry. Plasma …
and is used to characterize the mitochondria‐dependent pathway of this type of cell death.
Techniques currently used to measure cytochrome c release, Western blot and fluorescence
microscopy of immunolabeled cells, are time‐consuming and inaccurate, and the latter is
still limited by sample size. Methods: We developed a rapid and reliable technique to detect
cytochrome c release during drug‐induced apoptosis, using flow cytometry. Plasma …
Background
Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria‐dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time‐consuming and inaccurate, and the latter is still limited by sample size.
Methods
We developed a rapid and reliable technique to detect cytochrome c release during drug‐induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL‐60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells.
Results
The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4–8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c.
Conclusions
This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose. © 2006 International Society for Analytical Cytology
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