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Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D

Nature Protocols volume 2, pages 187–190 (2007)Cite this article

Abstract

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.

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Figure 1: Data obtained using the current staining method on human thymocytes.
Figure 2: Staining with Hoechst 33342 (HO342), 7-amino-actinomycin D (7-AAD), and detection of cell surface immunofluorescence.

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Acknowledgements

The authors thank the late Dr. Janis V. Giorgi for her contributions to the development of the original method (Schmid et al., Cytometry 15(12), 1994). This work was supported by National Institute of Health awards CA-16042 and AI-28697.

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Authors and Affiliations

  1. Department of Hematology/Oncology, Los Angeles, 90095, California, USA

    Ingrid Schmid & Beth D Jamieson

  2. Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, 90095, California, USA

    Christel Uittenbogaart

  3. Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, 90095, California, USA

    Christel Uittenbogaart

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  1. Ingrid Schmid
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  3. Beth D Jamieson
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Correspondence to Ingrid Schmid.

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Schmid, I., Uittenbogaart, C. & Jamieson, B. Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D. Nat Protoc 2, 187–190 (2007). https://doi.org/10.1038/nprot.2006.458

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