Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

Pellicciari, C; Mangiarotti, R; Bottone, M G; Danova, M; Wang, E

doi:10.1002/cyto.990210404

Title: Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

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Abstract

Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNAmore »« less

Authors:

Pellicciari, C; Mangiarotti, R; Bottone, M G; Danova, M ^[1]; Wang, E ^[2]

Univ. of Pavia (Italy)
Jewish General Hospital, Montreal, Quebec (Canada)

Publication Date:: Fri Dec 01 00:00:00 EST 1995

Sponsoring Org.:: USDOE

OSTI Identifier:: 456822

Resource Type:: Journal Article

Journal Name:: Cytometry

Additional Journal Information:: Journal Volume: 21; Journal Issue: 4; Other Information: PBD: 1 Dec 1995

Country of Publication:: United States

Language:: English

Subject:: 55 BIOLOGY AND MEDICINE, BASIC STUDIES; 44 INSTRUMENTATION, INCLUDING NUCLEAR AND PARTICLE DETECTORS; CELL FLOW SYSTEMS; EVALUATION; DNA; QUANTITATIVE CHEMICAL ANALYSIS; FIBROBLASTS; CELL PROLIFERATION; PROTEINS; DETECTION; IMMUNOASSAY; FLUORESCEIN; MONOCLONAL ANTIBODIES; BUDR; BIOLOGICAL MARKERS

Citation Formats


                    Pellicciari, C, Mangiarotti, R, Bottone, M G, Danova, M, and Wang, E. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content.  United States: N. p., 1995. 
        Web.  doi:10.1002/cyto.990210404.

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                    Pellicciari, C, Mangiarotti, R, Bottone, M G, Danova, M, & Wang, E. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content.  United States.  https://doi.org/10.1002/cyto.990210404

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                    Pellicciari, C, Mangiarotti, R, Bottone, M G, Danova, M, and Wang, E. 1995.  
        "Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content".  United States.  https://doi.org/10.1002/cyto.990210404.

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@article{osti_456822,

  title        = {Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content},

  author       = {Pellicciari, C and Mangiarotti, R and Bottone, M G and Danova, M and Wang, E},

  abstractNote = {Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G{sub 0} (statin positive) cells can be discriminated from the potentially cycling (statin negative) G{sub 1} cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G{sub 0}-G{sub 1} transition in unperturbed and drug-treated cell populations. 48 refs., 5 figs., 1 tab.},

  doi          = {10.1002/cyto.990210404},

  url          = {https://www.osti.gov/biblio/456822},
  journal      = {Cytometry},
number       = 4,

  volume       = 21,

  place        = {United States},

  year         = {Fri Dec 01 00:00:00 EST 1995},

  month        = {Fri Dec 01 00:00:00 EST 1995}

}

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