A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells

Belloc, Francis; Dumain, Patrice; Boisseau, Michel R.; Jalloustre, Claudine; Reiffers, Josy; Bernard, Philippe; Lacombe, Francis

doi:10.1002/cyto.990170108

Cited by 250Cell death mechanisms and regulationCell Image Analysis TechniquesMicrofluidic and Bio-sensing Technologies

A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells

Belloc, Francis, Dumain, Patrice, Boisseau, Michel R., Jalloustre, Claudine, Reiffers, Josy, Bernard, Philippe, Lacombe, Francis·1994·Cytometry

DOI 10.1002/cyto.990170108

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Abstract

A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells. © 1994 Wiley‐Liss, Inc.

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Total DNA intensity is an indicator of cell cycle phase.
“Total DNA intensity is an indicator of cell cycle phase that is regularly measured in both flow cytometry29 and HCS cell proliferation assays17.”
From A statistical framework for high-content phenotypic profiling using cellular feature distributions (2022) · cites this paper as [29]

Cell Death: The Significance of Apoptosis
Wyllie, A. H., Kerr, J. F. R., & Currie, A. R. (1980) · International review of cytology · doi:10.1016/s0074-7696(08)62312-8 · Cited by 7,235 · see citing works
A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry
Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., & Riccardi, C. (1991) · Journal of Immunological Methods · doi:10.1016/0022-1759(91)90198-o · Cited by 4,580 · see citing works
Features of apoptotic cells measured by flow cytometry
Darżynkiewicz, Z., Bruno, S., Bino, G. D., Gorczyca, W., Hotz, M. A., Lassota, P., & Traganos, F. (1992) · Cytometry · doi:10.1002/cyto.990130802 · Cited by 1,885 · see citing works
Apoptosis: Mechanisms and Roles in Pathology
Arends, M. J., & Wyllie, A. H. (1991) · International review of experimental pathology · doi:10.1016/b978-0-12-364932-4.50010-1 · Cited by 1,334 · see citing works
Apoptosis: The Biochemistry and Molecular Biology of Programmed Cell Death*
Schwartzman, R. A., & Cidlowski, J. A. (1993) · Endocrine Reviews · doi:10.1210/edrv-14-2-133 · Cited by 1,041 · see citing works
Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.
Gorczyca, W. A., GONG, J., & Darżynkiewicz, Z. (1993) · PubMed · Cited by 993 · see citing works
Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia
Barry, M. A., Behnke, C. A., & Eastman, A. R. (1990) · Biochemical Pharmacology · doi:10.1016/0006-2952(90)90733-2 · Cited by 876 · see citing works
Apoptosis induced by anticancer drugs
Hickman, J. A. (1992) · Cancer and Metastasis Reviews · doi:10.1007/bf00048059 · Cited by 860 · see citing works
Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis.
Savill, J., Hogg, N., Ren, Y., & Haslett, C. (1992) · Journal of Clinical Investigation · doi:10.1172/jci116019 · Cited by 813 · see citing works
Apoptosis and disease
Carson, D. A., & Ribeiro, J. M. (1993) · The Lancet · doi:10.1016/0140-6736(93)91154-e · Cited by 684 · see citing works
Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation
Cohen, G. M., Sun, X. M., Snowden, R. T., Dinsdale, D. D., & Skilleter, D. N. (1992) · Biochemical Journal · doi:10.1042/bj2860331 · Cited by 672 · see citing works
Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview
Wyllie, A. H. (1992) · Cancer and Metastasis Reviews · doi:10.1007/bf00048057 · Cited by 588 · see citing works
Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry
Dive, C., Gregory, C. D., Phipps, D. J., et al. (1992) · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research · doi:10.1016/0167-4889(92)90048-g · Cited by 544 · see citing works
Internucleosomal DNA Cleavage Should not be the Sole Criterion for Identifying Apoptosis
Collins, R. J., HARMON, B. V., Gobe, G. C., et al. (1992) · International Journal of Radiation Biology · doi:10.1080/09553009214551201 · Cited by 458 · see citing works
Apoptosis: molecular mechanisms in programmed cell death.
Fésüs, L., Davies, P. J., & Piacentini, M. (1991) · PubMed · Cited by 423 · see citing works
HL-60 cells induced to differentiate towards neutrophils subsequently die via apoptosis
Martin, S. J., BRADLEY, J. G., & Cotter, T. G. (1990) · Clinical & Experimental Immunology · doi:10.1111/j.1365-2249.1990.tb08110.x · Cited by 381 · see citing works
Comparative evaluation of several DNA binding dyes in the detection of apoptosis‐associated chromatin degradation by flow cytometry
Telford, W. G., King, L. E., & Fraker, P. J. (1992) · Cytometry · doi:10.1002/cyto.990130205 · Cited by 374 · see citing works
Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death
Brach, M., deVos, S., Gruss, H., & Herrmann, F. (1992) · Blood · doi:10.1182/blood.v80.11.2920.2920 · Cited by 359 · see citing works
Resolution of acute inflammation and the role of apoptosis in the tissue fate of granulocytes
Haslett, C. (1992) · Clinical Science · doi:10.1042/cs0830639 · Cited by 328 · see citing works
Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells.
Bhalla, K. N., Ibrado, A. M., Tourkina, E., Tang, C., Mahoney, M. E., & Huang, Y. (1993) · PubMed · Cited by 273 · see citing works
Induction of apoptotic cell death in chronic lymphocytic leukemia by 2- chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine
Robertson, L. E., Chubb, S., Meyn, R., et al. (1993) · Blood · doi:10.1182/blood.v81.1.143.bloodjournal811143 · Cited by 273 · see citing works
Hormone-regulated apoptosis results from reentry of differentiated prostate cells onto a defective cell cycle.
Colombel, M. C., Olsson, C. A., Ng, P., et al. (1992) · PubMed · Cited by 242 · see citing works
Induction of apoptotic cell death in chronic lymphocytic leukemia by 2- chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine
Robertson, L. E., Chubb, S., Meyn, R., et al. (1993) · Blood · doi:10.1182/blood.v81.1.143.143 · Cited by 231 · see citing works
Delayed internucleosomal DNA fragmentation in programmed cell death
Zakeri, Z. F., Quaglino, D., Latham, T. E., et al. (1993) · The FASEB Journal · doi:10.1096/fasebj.7.5.8462789 · Cited by 222 · see citing works
Apoptosis in interleukin-3-dependent haemopoietic cells
Ormerod, M. G., Collins, M. K., Rodríguez‐Tarduchy, G., et al. (1992) · Journal of Immunological Methods · doi:10.1016/0022-1759(92)90305-d · Cited by 204 · see citing works
Increased membrane permeability of apoptotic thymocytes: A flow cytometric study
Ormerod, M. G., Sun, X., Snowden, R. T., et al. (1993) · Cytometry · doi:10.1002/cyto.990140603 · Cited by 200 · see citing works
Introduction to Flow Cytometry
Watson, J. E. M. (1991) · Cambridge University Press eBooks · doi:10.1017/cbo9780511565090 · Cited by 196 · see citing works
Cell Cycle Analysis of p53-Induced Cell Death in Murine Erythroleukemia Cells
Ryan, J. J., Danish, R., Gottlieb, C. A., & Clarke, M. F. (1993) · Molecular and Cellular Biology · doi:10.1128/mcb.13.1.711 · Cited by 196 · see citing works
Induction of internucleosomal DNA fragmentation in human myeloid leukemia cells by 1-beta-D-arabinofuranosylcytosine.
Gunji, H., Kharbanda, S. M., & Kufe, D. W. (1991) · PubMed · Cited by 193 · see citing works
APO‐1 mediated apoptosis or proliferation in human chronic B lymphocytic leukemia: Correlation with bcl‐2 oncogene expression
Mapara, M. Y., Bargou, R. C., Zugck, C., et al. (1993) · European Journal of Immunology · doi:10.1002/eji.1830230320 · Cited by 176 · see citing works
Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase I and II inhibitors
Solary, É., Bertrand, R., Kohn, K. W., & Pommier, Y. G. (1993) · Blood · doi:10.1182/blood.v81.5.1359.1359 · Cited by 169 · see citing works
Bcl-2: an antidote to programmed cell death.
Korsmeyer, S. (1992) · PubMed · Cited by 153 · see citing works
Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-α
Takeda, Y., Watanabe, H. K., Yonehara, S., Yamashita, T., Salto, S., & Sendo, F. (1993) · International Immunology · doi:10.1093/intimm/5.6.691 · Cited by 151 · see citing works
Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells.
Lalande, M., Ling, V. W. T., & Miller, R. G. (1981) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.78.1.363 · Cited by 150 · see citing works
The cell cycle and cell death
Rubin, L. L., Philpott, K. L., & Brooks, S. F. (1993) · Current Biology · doi:10.1016/0960-9822(93)90211-6 · Cited by 132 · see citing works
A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes
Sun, X., Snowden, R. T., Skilleter, D. N., et al. (1992) · Analytical Biochemistry · doi:10.1016/0003-2697(92)90251-2 · Cited by 128 · see citing works
Radiation-induced apoptosis in a murine T-cell hybridoma.
Warters, R. L. (1992) · PubMed · Cited by 112 · see citing works
Overexpressed Csk Tyrosine Kinase Is Localized in Focal Adhesions, Causes Reorganization of αvβ5 Integrin, and Interferes with Hela Cell Spreading
Bergman, M., Joukov, V., Virtanen, I., et al. (1995) · Molecular and Cellular Biology · doi:10.1128/mcb.15.2.711 · Cited by 110 · see citing works
The involvement of oncogenes and tumor suppressor genes in the control of apoptosis
Oren, M. (1992) · Cancer and Metastasis Reviews · doi:10.1007/bf00048060 · Cited by 96 · see citing works
Regulation of T lymphocyte apoptosis. Signals for the antagonism between activation- and glucocorticoid-induced death
Iseki, R., Mukai, M., & Iwata, M. (1991) · The Journal of Immunology · doi:10.4049/jimmunol.147.12.4286 · Cited by 93 · see citing works
Apoptosis or Programmed Cell Death
Waring, P., Kos, F. J., & Müllbacher, A. (1991) · Medicinal Research Reviews · doi:10.1002/j.1098-1128.1991.tb00004.x · Cited by 78 · see citing works
Vitality measurement using spectrum shift in Hoechst 33342 stained cells
Ellwart, J. W., & Dormer, P. G. (1990) · Cytometry · doi:10.1002/cyto.990110204 · Cited by 62 · see citing works
A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation
Hardin, J. A., Sherr, D. H., DeMaria, M., et al. (1992) · Journal of Immunological Methods · doi:10.1016/0022-1759(92)90217-h · Cited by 58 · see citing works
Cell Cycle Analysis of p53-Induced Cell Death in Murine Erythroleukemia Cells
Ryan, J. J., Danish, R., Gottlieb, C. A., et al. (1993) · Molecular and Cellular Biology · doi:10.1128/mcb.13.1.711-719.1993 · Cited by 53 · see citing works
Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342.
Loken, M. R. (1980) · Journal of Histochemistry & Cytochemistry · doi:10.1177/28.1.6153191 · Cited by 51 · see citing works
Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting.
Mg, P., Lj, S., Dean, P. N., et al. (1985) · PubMed · Cited by 34 · see citing works
Flow cytometry analysis of DNA degradation in thymocytes of gamma-irradiated or hydrocortisone treated rats.
Pechatnikov, V. A., Afanasyev, V. N., Korol', B. A., et al. (1986) · PubMed · Cited by 12 · see citing works

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Authors: Belloc, F., Dumain, P., Boisseau, M. R., Jalloustre, C., Reiffers, J., Bernard, P., & Lacombe, F.
Affiliations: Centre National de la Recherche Scientifique (FR)
ORCID: Lacombe, Francis
Venue: Cytometry
Publisher: Wiley
Published: 1994-09
Volume: 17
Issue: 1
Pages: 59-65
ISSN: 0196-4763 (ISSN-L)
1097-0320
DOI: 10.1002/cyto.990170108
OpenAlex: W2015181708
Cited by: 250
References: 47
Citations by year: 2026: 5 · 2025: 6 · 2024: 2 · 2023: 2 · 2022: 3 · 2021: 10 · 2020: 7 · 2019: 7 · 2018: 7 · 2017: 10 · 2016: 16 · 2015: 7 · 2014: 11 · 2013: 13 · 2012: 5
FWCI: 4.05
Type: journal article
Language: en
Open access: closed
Licence: onlinelibrary.wiley.com/termsAndConditions#vor (vor, 3841 d)
Locations: Cytometry (publishedVersion)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Bio-sensing Technologies
Keywords: apoptosis detection, Hoechst 33342, propidium iodide, cell cycle analysis, chromatin condensation, necrosis, polymorphonuclear leukocytes, U937 cells, cell sorting, DNA degradation
MeSH terms: Antineoplastic Agents, Benzimidazoles, Cell Cycle, Cell Membrane, Cells, Cultured, Chromatin, DNA Damage, DNA, Neoplasm, Electrophoresis, Agar Gel, Flow Cytometry, Fluorescent Dyes, Humans, Monocytes, Neutrophils, Propidium, Staining and Labeling, Tumor Cells, Cultured, Leukemia, Promyelocytic, Acute, Apoptosis

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A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells

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