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 Previous Article | Table of Contents | Next Article 
Annexin V for flow cytometric detection of phosphatidylserine expression on
B cells undergoing apoptosis
G Koopman, CP Reutelingsperger, GA Kuijten, RM Keehnen, ST Pals and MH van Oers
Department of Pathology, Academic Medical Center, Amsterdam, The
Netherlands.
Apoptosis, or programmed cell death, is a general mechanism for removal of
unwanted cells from the immune system. It is characterized by chromatin
condensation, a reduction in cell volume, and endonuclease cleavage of DNA
into oligonucleosomal length fragments. Apoptosis is also accompanied by a
loss of membrane phospholipid asymmetry, resulting in the exposure of
phosphatidylserine at the surface of the cell. Expression of
phosphatidylserine at the cell surface plays an important role in the
recognition and removal of apoptotic cells by macrophages. Here we describe
a new method for the detection of apoptotic cells by flow cytometry, using
the binding of fluorescein isothiocyanate-labeled annexin V to
phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated
germinal center B cells are cultured under apoptosis inducing conditions,
all cells showing chromatin condensation strongly stain with annexin V,
whereas normal cells are annexin V negative. Moreover, DNA fragmentation is
only found in the annexin V-positive cells. The nonvital dye ethidium
bromide was found to stain a subpopulation of the annexin V-positive
apoptotic cells, increasing with time. Our results indicate that the phase
in apoptosis that is characterized by chromatin condensation coincides with
phosphatidylserine exposure. Importantly, it precedes membrane damage that
might lead to release from the cells of enzymes that are harmful to the
surrounding tissues. Annexin V may prove important in further unravelling
the regulation of apoptosis.
Volume 84,
Issue 5,
pp. 1415-1420,
09/01/1994
Copyright © 1994 by The American Society of Hematology
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