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Quantitative immunofluorescence in single Saccharomyces cerevisiae cells.
P D Eitzman1, J L Hendrick, F Srienc
1Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108.
Cytometry
|July 1, 1989
Summary
A new staining method enables simultaneous measurement of DNA and beta-galactosidase antigen in yeast using flow cytometry. This technique accurately quanties enzyme levels at the single-cell level.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Simultaneous intracellular analysis of DNA and protein is crucial for understanding cellular processes.
- Traditional methods often require multiple steps and may not provide single-cell resolution.
- Developing efficient techniques for yeast cell analysis is essential for genetic and biochemical studies.
Purpose of the Study:
- To develop and optimize a staining procedure for simultaneous determination of intracellular DNA and a specific antigen (beta-galactosidase) in Saccharomyces cerevisiae.
- To validate the accuracy of the developed method by correlating immunofluorescence with enzyme activity.
- To enable single-cell analysis of antigen expression in relation to cell cycle position.
Main Methods:
- Development of a novel staining protocol for yeast cells.
- Optimization of cell fixation and cell wall removal for enhanced immunofluorescence.
- Indirect immunofluorescence staining for detecting beta-galactosidase antigen.
- Single-laser flow cytometry for simultaneous measurement of DNA content and immunofluorescence.
- Enzyme activity assays using o-nitrophenyl-beta-D-galactopyranoside.
Main Results:
- Successful simultaneous determination of intracellular DNA and beta-galactosidase antigen in yeast.
- Optimized cell preparation procedures minimized cell loss and maximized immunofluorescence signals.
- High correlation observed between average immunofluorescence levels and average beta-galactosidase activity per cell.
- Demonstrated accuracy of the method at the single-cell level, holding true for varying gene copy numbers.
- Method proved adaptable for detecting other antigens by altering primary antibodies.
Conclusions:
- The developed staining procedure offers a robust and efficient method for simultaneous DNA and antigen quantification in yeast.
- This technique facilitates detailed investigation of enzyme expression dynamics across the cell cycle.
- The adaptable protocol has broad potential for analyzing various cellular components in Saccharomyces cerevisiae and potentially other organisms.