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Apoptosis is a programmed series of events that leads to the removal of old, unwanted, or damaged cells from the tissues of multicellular organisms [1]. In contrast to necrotic cell death, which is characterized by loss of membrane potential and the release of cellular contents into the intercellular space, apoptosis is a “clean” death distinguished by the controlled demolition of the cell. During apoptosis the cell is dismantled from within and the cellular contents are dispersed in small membrane bound vesicles which are then taken up by phagocytic cells. This organized dispersal and recycling of the cellular contents prevents the type of local inflammation and damage to surrounding cells associated with necrotic cell death [2]. Apoptosis is critical to the proper development of multicellular organisms and for maintenance of homeostasis in the mature organism. Defects in apoptotic programming are linked to major diseases such as cancer, neurodegenerative diseases, and heart disease [3-6].

    Caspases are a family of cysteine proteases that are central to the apoptotic pathway, acting as both signaling molecules and the end effectors of the cell death program [7]. The family is divided into two groups, initiator caspases and effector caspases. The initiator caspases are mediators of apoptotic signaling whereas the effectors are responsible for degradation of the cellular contents. Caspase 3 is the most well studied of the effector caspases and its activation is considered one of the hallmarks of apoptotic cell death. Caspases are transcribed as precursor enzymes or pro-caspases which are activated by proteolytic cleavage. Several manufacturers have produced antibodies which only recognize the active or cleaved form of caspase 3, allowing for identification of apoptosis activation in cell culture and tissue sections by immunofluorescence. Here we demonstrate how it is possible to quantify the level of activated caspase 3 on a cell by cell basis. using CyteSeer's Protein Expression algorithm.

Representative images of control cells (A-C) and cells treated with 1µM staurosporine (D-F) are shown. Cells were identified by the DAPI signal (A&D). Control cells showed very low levels of staining for activated caspase 3 (B), while treatment with 1µM staurosporine produced robust activated caspase 3 staining (E). CyteSeer® applies a mask to areas of the image it recognizes as staining positive for activated Caspase 3 (C,F).

Increasing doses of staurosporine over a three hour incubation result in increased activation of caspase 3 as determined by CyteSeer® using the protein expression algorithm to analyze caspase 3 staining in the experimental images. Each treatment group represents eight wells in a 96 well plate with 400-500 cells analyzed per well. Analysis using Cyteseer® took, on average, less than 5 minutes to complete.

    To initiate the intrinsic apoptotic pathway, HeLa cells plated in a 96 well optical plate were treated with varying concentrations of staurosporine (30nM-1µM) for three hours while control cells were treated with vehicle alone (DMSO). Following the incubation time cells were fixed and stained for activated caspase 3 (Cell Signaling Cat# 9661); DAPI stain was used to identify the nuclei. The plates were then imaged using an automated microscope (Beckman Coulter IC100) and image analysis was performed using Cyteseer’s Protein Expression Algorithm (Figure 1). Treatment with staurosporine resulted in activation of caspase 3 in a dose responsive manner (Figure 2). The data provide a Z’ statistic of 0.506 suggesting that measurement of caspase 3 activation using CyteSeer® could be used as an excellent screening assay with application in cytotoxicity monitoring, the identification of anti-cancer compounds, or the identification of anti-apoptotic molecules.

The Protein Expression algorithm is just one of the many robust analysis tools contained within Cyteseer®. Here we demonstrated that the Protein Expression algorithm can be applied to apoptosis research in the analysis of caspase 3 activation. In these experiments we used a 96 well plate format and automated microscopy, however, CyteSeer is flexible and can perform similar comparative analyses on a few images collected from individual slides or can be applied to screens in larger plate formats. The excellent Z’ score achieved in these experiments suggests that staining for active caspase 3 could readily be applied to larger screens of compound libraries for cytotoxicity, anti-cancer, or anti-apoptotic properties.

1.    Taylor, R.C., S.P. Cullen, and S.J. Martin, Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol, 2008. 9(3): p. 231-41.
2.    Rock, K.L. and H. Kono, The inflammatory response to cell death. Annu Rev Pathol, 2008. 3: p. 99-126.
3.    Eckert, A., et al., Increased apoptotic cell death in sporadic and genetic Alzheimer's disease. Ann N Y Acad Sci, 2003. 1010: p. 604-9.
4.    Faubel, S. and C.L. Edelstein, Caspases as drug targets in ischemic organ injury. Curr Drug Targets Immune Endocr Metabol Disord, 2005. 5(3): p. 269-87.
5.    Hamacher-Brady, A., N.R. Brady, and R.A. Gottlieb, The interplay between pro-death and pro-survival signaling pathways in myocardial ischemia/reperfusion injury: apoptosis meets autophagy. Cardiovasc Drugs Ther, 2006. 20(6): p. 445-62.
6.    Hunter, A.M., E.C. LaCasse, and R.G. Korneluk, The inhibitors of apoptosis (IAPs) as cancer targets. Apoptosis, 2007. 12(9): p. 1543-68.
7.    Li, J. and J. Yuan, Caspases in apoptosis and beyond. Oncogene, 2008. 27(48): p. 6194-206.

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