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A stable propidium iodide staining procedure for flow cytometry
Abstract:
A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.
Insights
This study details a simple propidium iodide staining method for DNA analysis in various cells. Optimized RNAse incubation and storage conditions ensure stable, reliable flow cytometry results over time.
Area of Science:
- Cell Biology
- Biotechnology
- Analytical Chemistry
Background:
- Accurate DNA content analysis via flow cytometry is crucial for biological research.
- Standard propidium iodide (PI) staining protocols require optimization for RNA removal and sample stability.
Purpose of the Study:
- To describe and validate a robust propidium iodide (PI) staining procedure for DNA analysis.
- To establish optimal conditions for RNAse digestion and sample storage for flow cytometry.
Main Methods:
- Utilized propidium iodide (PI) staining with RNAse treatment for DNA-specific fluorescence.
- Investigated varying RNAse concentrations and incubation times on murine erythroleukemia cells (MELC).
- Assessed sample stability under different storage conditions (refrigeration, freezing) and with preservatives.
Main Results:
- Achieved specific nuclear DNA staining by enzymatic RNA removal.
- Identified optimal RNAse incubation times (e.g., 30 min at 37°C for 1 mg/ml RNAse) for consistent histograms.
- Demonstrated long-term stability of stained unfixed cells for over 2 weeks at 4°C, and up to 1-2 months with preservatives like sodium azide and glycerol.
Conclusions:
- The described PI staining method is simple, reproducible, and yields stable results.
- Optimized RNAse digestion and storage protocols enhance reliability for comparative flow cytometry.
- This procedure is suitable for long-term, repetitive sampling and analysis of cell populations.