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A simple quantitative method of estimation of cell-intactness based on ethidium bromide fluorescence
1Indian Institute of Chemical Biology, Jadavpur, Calcutta.
Biochemistry International
|August 1, 1988
Summary
A new ethidium bromide (EB) fluorescence method accurately assesses mammalian cell plasma membrane integrity. This rapid technique reliably detects damaged cells, offering a simpler alternative to traditional enzyme-based assays.
Area of Science:
- Cell Biology
- Biochemistry
- Biophysics
Background:
- Assessing plasma membrane intactness is crucial for cell viability studies.
- Conventional methods like marker enzyme assays can be time-consuming and less sensitive.
- A need exists for rapid, reliable, and sensitive methods to quantify cellular membrane integrity.
Purpose of the Study:
- To develop and validate a quantitative fluorescence-based method for estimating plasma membrane intactness in mammalian cells.
- To compare the efficacy of the new method with established techniques.
Main Methods:
- Utilized ethidium bromide (EB) fluorescence, which binds to DNA in cells with compromised membranes.
- Measured fluorescence intensity (excitation-emission 365-580 nm) in goat epididymal spermatozoa.
- Corrected for non-specific fluorescence and compared readings to sonicated (fully damaged) cells.
Main Results:
- The EB-fluorescence method demonstrated high accuracy, yielding intactness values of 99.5 +/- 0.3%.
- Results closely correlated with the conventional marker enzyme method (97 +/- 0.8%).
- The method is highly sensitive, detecting as low as 0.5% damaged or 'leaky' cells.
Conclusions:
- The ethidium bromide fluorescence method provides a simpler, faster, and more reliable assessment of cellular plasma membrane intactness.
- This technique is suitable for quantifying membrane integrity in cell preparations, including spermatozoa.
- The method's high sensitivity and accuracy make it a valuable tool in cell biology research.