Nucleic acid dyes for detection of apoptosis in live cells

Abstract

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-GI DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4my-l dim thymocytes were included in the dye dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin.However, the dyes mo-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser. 0 1995 Wiley-Liss, Inc.Key terms: LDS-751, thymocytes, programmed cell death, Apoptosis is a mode of cell death important in a wide variety of situations (5,2054). It is characterized by nuclear chromatin condensation and morphological changes that occur in the absence of plasma membrane breakdown. The nuclear DNA of apoptotic cells is also nicked, although not all model systems show the extensive DNA degradation seen in cells of hematopoietic lineages (4,6,35). Many of the other characteristics of apoptotic cells have been noted in some, but not all, models studied.The differences in the model systems studied has led to the appearance of several different assays for apoptosis (31). Electron microscopic examination is usually considered the most definitive assay. Other less cumbersome assays have been widely used. These include gel electrophoresis to detect the laddering characteristic of internucleosomal cleavage of DNA, flow cytometric detection of cells with "sub-G," DNA content, and various flow cytometric and in situ nick extension assays for the detection of DNA strand breaks (10,17). A potential limitation of all of these assays is that the cells are no longer viable.Some reported assays have been capable of detecting apoptosis in live cells. The DNA dye Hoechst 33342 is more rapidly taken up by apoptotic thymocytes (12,15,18,26). This has been attributed to an increase in membrane permeability. This assay is simple but requires W excitation. An increase in efflux of fluorescein after loading with fluorescein diacetate (26) and an increased influx rate for ethidium bromide (22) have been attributed to the same increased membrane permeability. In a second type of assay, a decrease in cell surface antigens is used to identify apoptotic cells. Antigens studied have included the CD45 antigen ...

An improved flow cytometric assay for detection and discrimination between malignant cells and atypical mesothelial cells, in serous cavity effusions

“…SYTO-16 and 7-AAD were used in the initial panel for the analysis of nucleated cells and the exclusion of apoptotic cells, necrotic cells, and debris (27)(28)(29)(30). In this way, the possibility of false positive clusters in CD45À/ CD71þ gating, was excluded, also eliminating the possibility of compensation errors and making more reliable determinations of small clusters (at least 0.1% of nucleated cells).…”

Changes in L-selectin expression on CD34-positive cells upon cryopreservation of peripheral blood stem cell transplants

“…Staining of incubated samples with the viability stain 7-AAD revealed that the population with the low forward scatter (population 1) was partly 7-AAD positive (non-viable) but also partly 7-AAD negative, while the population with the high forward scatter (population 2) was 7-AAD negative. Parallel experiments in our laboratory in which Syto 16 was used as an apoptosis marker 22,23 together with 7-AAD staining showed that the part of population 1 with the low FSC that was 7-AAD negative had a relatively low Syto 16 staining which is indicative for early apoptosis. Population 2 is considered to be relevant for clinical recovery and varied largely between individual samples.…”

Pharmacological properties of a pore induced by raising intracellular Ca2+

“…5C). SYTO 13 (a dye shown to discriminate apoptotic, live, and dead cells) was used to observe whether the cell population permeabilized by ionomycin would be viable, as indicated elsewhere (32,78). PI and SYTO 13 were used at the same time, as SYTO 13 modified the peritoneal macrophage morphology after 2 min of incubation.…”