TO-PRO-3 iodide (TP3), a monomeric cyanine nucleic acid stain with a peak absorbance at 642nm and emission at 661nm, is best excited by a helium-neon (HeNe) laser (633nm). It was tested in monocytes and different cell lines under conditions of different fixatives, dye concentrations, labeling kinetics and RNAse concentrations for mono-, bi- and tri-parametric flow cytometric cell cycle analysis to establish the best protocol for DNA analysis in terms of G(1) peak CV, G(2)/G(1) ratio and minimal amount of debris. A linear increase in G(1) peak position was found from 0.1 to 2muM TP3 concentrations. Fixatives 70% ethanol or 1% methanol-free formaldehyde, followed by 70% ethanol, resulted in the best DNA histograms. Although different protocols were found to be cell-type specific, in general, excellent results were obtained with 30min incubation with 0.5muM TP3 plus RNAse in almost all cell lines tested. These data show that TP3 is an alternative method to propidium iodide (PI), the most commonly used DNA-specific probe in flow cytometry. The most important advantage of using TP3 in combination with other fluorochromes, such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in bi- or tri-parametric flow cytometric analysis, is that there is no need for fluorescence compensation for the TP3 signals.
Multi-parametric flow cytometric cell cycle analysis using TO-PRO-3 iodide (TP3): Detailed protocols
Mazzini G;
2008
Abstract
TO-PRO-3 iodide (TP3), a monomeric cyanine nucleic acid stain with a peak absorbance at 642nm and emission at 661nm, is best excited by a helium-neon (HeNe) laser (633nm). It was tested in monocytes and different cell lines under conditions of different fixatives, dye concentrations, labeling kinetics and RNAse concentrations for mono-, bi- and tri-parametric flow cytometric cell cycle analysis to establish the best protocol for DNA analysis in terms of G(1) peak CV, G(2)/G(1) ratio and minimal amount of debris. A linear increase in G(1) peak position was found from 0.1 to 2muM TP3 concentrations. Fixatives 70% ethanol or 1% methanol-free formaldehyde, followed by 70% ethanol, resulted in the best DNA histograms. Although different protocols were found to be cell-type specific, in general, excellent results were obtained with 30min incubation with 0.5muM TP3 plus RNAse in almost all cell lines tested. These data show that TP3 is an alternative method to propidium iodide (PI), the most commonly used DNA-specific probe in flow cytometry. The most important advantage of using TP3 in combination with other fluorochromes, such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in bi- or tri-parametric flow cytometric analysis, is that there is no need for fluorescence compensation for the TP3 signals.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
