Advanced

Multi-parametric flow cytometric cell cycle analysis using TO-PRO-3 iodide (TP3) : detailed protocols

Tavecchio, Michele LU ; Simone, Matteo; Bernasconi, Sergio; Tognon, Gianluca; Mazzini, Giuliano and Erba, Eugenio (2008) In Acta Histochemica 110(3). p.44-232
Abstract

TO-PRO-3 iodide (TP3), a monomeric cyanine nucleic acid stain with a peak absorbance at 642 nm and emission at 661 nm, 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 G1 peak CV, G2/G1 ratio and minimal amount of debris. A linear increase in G1 peak position was found from 0.1 to 2 microM TP3 concentrations. Fixatives 70% ethanol or 1% methanol-free formaldehyde, followed by 70% ethanol, resulted in the best DNA histograms. Although different... (More)

TO-PRO-3 iodide (TP3), a monomeric cyanine nucleic acid stain with a peak absorbance at 642 nm and emission at 661 nm, 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 G1 peak CV, G2/G1 ratio and minimal amount of debris. A linear increase in G1 peak position was found from 0.1 to 2 microM 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 30 min incubation with 0.5 microM 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.

(Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
keywords
Apoptosis, Carbocyanines, Cell Cycle, Cell Line, Tumor, Cells, Cultured, DNA, DNA Probes, Flow Cytometry, Fluorescent Dyes, G1 Phase, G2 Phase, HL-60 Cells, HT29 Cells, Humans, Jurkat Cells, Lasers, Microscopy, Fluorescence, Monocytes, Phycoerythrin, Propidium, Journal Article
in
Acta Histochemica
volume
110
issue
3
pages
44 - 232
publisher
Elsevier
external identifiers
  • scopus:43049086277
ISSN
0065-1281
DOI
10.1016/j.acthis.2007.10.007
language
English
LU publication?
no
id
cc005874-ca3c-48d7-a8cc-7a588d3da2bd
date added to LUP
2017-03-07 09:14:43
date last changed
2017-05-30 10:32:09
@article{cc005874-ca3c-48d7-a8cc-7a588d3da2bd,
  abstract     = {<p>TO-PRO-3 iodide (TP3), a monomeric cyanine nucleic acid stain with a peak absorbance at 642 nm and emission at 661 nm, 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 G1 peak CV, G2/G1 ratio and minimal amount of debris. A linear increase in G1 peak position was found from 0.1 to 2 microM 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 30 min incubation with 0.5 microM 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.</p>},
  author       = {Tavecchio, Michele and Simone, Matteo and Bernasconi, Sergio and Tognon, Gianluca and Mazzini, Giuliano and Erba, Eugenio},
  issn         = {0065-1281},
  keyword      = {Apoptosis,Carbocyanines,Cell Cycle,Cell Line, Tumor,Cells, Cultured,DNA,DNA Probes,Flow Cytometry,Fluorescent Dyes,G1 Phase,G2 Phase,HL-60 Cells,HT29 Cells,Humans,Jurkat Cells,Lasers,Microscopy, Fluorescence,Monocytes,Phycoerythrin,Propidium,Journal Article},
  language     = {eng},
  number       = {3},
  pages        = {44--232},
  publisher    = {Elsevier},
  series       = {Acta Histochemica},
  title        = {Multi-parametric flow cytometric cell cycle analysis using TO-PRO-3 iodide (TP3) : detailed protocols},
  url          = {http://dx.doi.org/10.1016/j.acthis.2007.10.007},
  volume       = {110},
  year         = {2008},
}