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Flow cytometry and staining kinetics to monitor the G0/G1 transition

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Methods in Cell Science

Abstract

The phenanthridine dyes propidium iodide (PI) and ethidium bromide (EB) have contributed to make DNA quantitation a routine measurement by flow cytometry. As planar molecules they easily intercalate between base pairs of double stranded nucleic acids. When the quantity of dye exceeds the amount of DNA, the intensity of emitted fluorescence can be related to the amount of DNA. This is, in fact, the basic requirement of quantitative measurement. When staining is performed at low dye concentration and there are no sufficient molecules to saturate all the available DNA-binding sites, the emitted fluorescence, other than the true DNA quantity, may be dependent on the state of chromatin condensation. A staining procedure aimed to stress the influence of nuclear structure on the emitted fluorescence of PI is described. This is achieved by means of a very low dye concentration (<0.01 μg/ml PI) to guarantee staining far below saturation condition. In this particular condition the staining rate slows down to be monitored by flow cytometry. As different experimental models the leucocytes of the normal human peripheral blood have been used taking into account the relatively condensed chromatin of granulocytes with respect to that of the mononuclear cells. Significantly higher fluorescence intensity have been obtained from mononuclear cells compared to that of the polymorphonuclear ones. Quiescent versus exponentially proliferating cell cultures had also been tested. In this staining condition low fluorescence intensity have been obtained by condensed chromatin structure (G0) in comparison to the more decondensed (G1) one.

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Abbreviations

PI:

propidium iodide

PBS:

phosphate buffered saline

BrdU:

bromodeoxyuridyne

FCM:

flow cytometry

PMNC:

polymorphonuclear cells

MNC:

mononuclear cells

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Authors and Affiliations

  1. Centro di Studio per l'Istochimica del Consiglio Nazionale delle Ricerche, Università di Pavia, Pavia, Italy

    Giuliano Mazzini, Roberta Alberici & Claudia Melchioni

  2. Dipartimento di Biologia Animale, Università di Pavia, Pavia, Italy

    Giuliano Mazzini, Roberta Alberici & Claudia Melchioni

  3. Medicina Interna e Oncologia Medica, Università e IRCCS Policlinico S. Matteo, Pavia, Italy

    Marco Danova

  4. Centro Istochimica, C.N.R., Piazza Botta 10, I-27100, Pavia, Italy

    Giuliano Mazzini

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  1. Giuliano Mazzini
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  2. Roberta Alberici
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  3. Claudia Melchioni
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  4. Marco Danova
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Mazzini, G., Alberici, R., Melchioni, C. et al. Flow cytometry and staining kinetics to monitor the G0/G1 transition. Methods Cell Sci 18, 165–177 (1996). https://doi.org/10.1007/BF00122167

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