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

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.

[1]  M. Loken.  Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342. , 1980, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[2]  P. Norris,et al.  A flow cytometric study of actinic reticuloid , 1989, Clinical and experimental dermatology.

[3]  Z. Darżynkiewicz,et al.  Difficulties and pitfalls in analysis of apoptosis. , 2001, Methods in cell biology.

[4]  C. Franceschi,et al.  Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level. , 1994, Experimental cell research.

[5]  M. Collins,et al.  Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. , 1992, Journal of immunological methods.

[6]  I Nicoletti,et al.  A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. , 1991, Journal of immunological methods.

[7]  Z. Darżynkiewicz,et al.  Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. , 2000, Experimental cell research.

[8]  J. Giorgi,et al.  Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability. , 1999, Cytometry.

[9]  Zbigniew Darzynkiewicz,et al.  Cytometric methods to detect apoptosis. , 2004, Methods in cell biology.

[10]  X. M. Sun,et al.  Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. , 1993, Cytometry.

[11]  I Schmid,et al.  Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. , 1992, Cytometry.

[12]  Z. Darżynkiewicz,et al.  Features of apoptotic cells measured by flow cytometry. , 1992, Cytometry.

[13]  I Schmid,et al.  A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. , 1994, Journal of immunological methods.

[14]  W. Telford,et al.  Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. , 1992, Cytometry.

[15]  M. Miller,et al.  Mercuric chloride induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction. , 1998, Toxicology and applied pharmacology.

[16]  P. Smith,et al.  Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells. , 1985, Experimental cell research.

[17]  D L Evans,et al.  Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. , 1992, Biochimica et biophysica acta.

[18]  C Haanen,et al.  A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. , 1995, Journal of immunological methods.

[19]  Z. Darżynkiewicz,et al.  Chapter 24 Difficulties and pitfalls in analysis of apoptosis , 2001 .

[20]  J. Giorgi,et al.  Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. , 1994, Cytometry.

[21]  Z. Darżynkiewicz,et al.  Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. , 1993, Cancer research.