A quantitative real-time approach for discriminating apoptosis and necrosis

Abstract

Apoptosis and necrosis are the two major forms of cell death mechanisms. Both forms of cell death are involved in several physiological and pathological conditions and also in the elimination of cancer cells following successful chemotherapy. Large number of cellular and biochemical assays have evolved to determine apoptosis or necrosis for qualitative and quantitative purposes. A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells. This highlights the essential requirement of an approach with a real-time analysis capability for discriminating the two forms of cell death. This paper describes a sensitive live cell-based method for distinguishing apoptosis and necrosis at single-cell level. The method uses cancer cells stably expressing genetically encoded FRET-based active caspase detection probe and DsRed fluorescent protein targeted to mitochondria. Caspase activation is visualized by loss of FRET upon cleavage of the FRET probe, while retention of mitochondrial fluorescence and loss of FRET probe before its cleavage confirms necrosis. The absence of cleavage as well as the retention of mitochondrial fluorescence indicates live cells. The method described here forms an extremely sensitive tool to visualize and quantify apoptosis and necrosis, which is adaptable for diverse microscopic, flow cytometric techniques and high-throughput imaging platforms with potential application in diverse areas of cell biology and oncology drug screening.

Polyphenolic Composition and Anti-Melanoma Activity of White Forsythia (Abeliophyllum distichum Nakai) Organ Extracts

“…Apoptosis and necrosis are highly conserved cell death mechanisms involved in the elimination of cancer cells by anti-cancer agents [ 17 ]. Distinguishing specific cell death subtypes is highly important to develop antineoplastic or other therapeutic drugs.…”

<p>K-3-Rh Protects Against Cerebral Ischemia/Reperfusion Injury by Anti-Apoptotic Effect Through PI3K-Akt Signaling Pathway in Rat</p>

“…Studies have found that myocardial cells have both necrotic and apoptotic forms during I/R in acute myocardial infarction, and apoptosis can be caused by acute myocardial infarction. 30,31 With the induction of external death signals, nucleated cells initiate their own internal gene expression and regulation through signal transduction pathways, leading to programmed process of cell death, which has a great effect…”

A high-throughput real-time in vitro assay using mitochondrial targeted roGFP for screening of drugs targeting mitochondria

“…Many innovative approaches were ascertained in developing Green fluorescent protein (GFP) based sensors for signaling molecules such as calcium, reactive oxygen species (ROS), ATP, pH, cAMP [11] , [12] , [13] , [14] , [15] . Genetically encoded fluorescent probe based cell death tools were also described for toxicological studies [16] , [17] , [18] , [19] . We have described a high-throughput FRET-based method for caspase profiling [16] , [20] .…”

“…Genetically encoded fluorescent probe based cell death tools were also described for toxicological studies [16] , [17] , [18] , [19] . We have described a high-throughput FRET-based method for caspase profiling [16] , [20] . Intracellular generation of ROS and dysfunctional redox signaling have been attributed to the cytotoxic activity of various antitumor agents.…”