Apoptosis assays

Apoptosis or programmed cell death is one of the regulatory mechanisms for the removal of unwanted cells. Apoptosis is induced by the stimulation of several different cell surface receptors in association with caspase activation. Apoptosis of a cell is thus a complicated process and can be assayed by various methods. Among widely used methods, the TUNEL assay is described here. 

TUNEL Assay (Terminal Transferase dUTP Nick End Labelling) 

5 units of TdT in its storage buffer (usually 0.5 ml) 0.25 nmol of FITC-dUTP (or BODIPY-dUTP) in its storage buffer Add distilled water to 50 ml. 

XTT assay is suitable for measuring cell proliferation, cell viability or cytotoxicity. The tetrazolium salts are converted into a coloured formazan product by cellular enzymes present in the mitochondria of a metabolically active cell. These enzymes are rapidly inactivated when a cell dies, and hence the activity of these enzymes can be used to monitor the viability of a cell. 

---

Apoptosis assay. ECRF24 or A2780 cells were seeded on 6-well plates (2 X 105 cells/ well) and grown 24 hours in complete medium before treatment. Compounds 1-3 were freshly dissolved in DMSO, diluted in complete medium and added to the cells at the final concentrations indicated in Table 2. After incubation for 72 h apoptosis was measured by flow cytometric determination of subdiploid cells after DNA extraction and subsequent staining with propidium iodide (PI) as described previously10. Briefly, cells were harvested and subsequently fixed in 70% ethanol at —20°C. After 2 h the cells were re-suspended in DNA extraction buffer (45 mM Na2HP04, 2.5 mM citric acid, and 1% Triton X-100, pH 7.4) for 20 min at 37°C. PI was added to a final concentration of 20 pg/mL and log scale red fluorescence was analyzed on a FACSCalibur (BD Biosciences, NJ, U.S.). 

Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations (http //probes.invitrogen.com/handbook/sections/0806.html). For instance, Vybrant Apoptosis Assay Kit 4 (Molecular Probes) detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide. 

C. Bernstein, H. Bernstein, H. Garewal, P. Dinning, R. Jabi, R. E. Sampliner, M. K. McCuskey, M. Panda, D. J. Roe, L. L Heureux and C. Payne, A bile-acid-induced apoptosis assay for colon cancer risk and associated quality control studies. Cancer Res., 1999, 59(10), 2353. 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

Bardales RH, Hailey LS, Xie SS, Schaefer RF, Hsu SM. In situ apoptosis assay for the detection of early acute myocardial infarction. Am J Pathol 1996 149 821-829. 

In addition to determining whether cells are alive or dead, it is often of interest to determine whether cell death was due to apoptosis. An apoptosis assay is the logical choice to screen libraries to identify candidate compounds for development of cancer therapeutics where inducing apoptosis is often the clinical goal. Screening to detect apoptosis is more likely to rule out problematic compounds that induce undesirable outcomes such as necrotic cell death. 

After a cell type (in vitro model system) has been chosen for cytotoxicity or apoptosis assays, several key parameters must be determined to characterize assay performance (1) optimal cell seeding density, (2) volume of culture medium per well, (3) equilibration period after dispensing cells, (4) concentration of compound to be tested, (5) length of exposure of cells to the test compound, and (6) selection of appropriate assay chemistry. 

One of the main challenges in designing cytotoxicity and apoptosis assays for screening is determining an appropriate length of compound exposure. Difficulties arise because different classes of... 

A detailed description of all methodologies and assays for detecting apoptosis is beyond the scope of this chapter. However, some of the most commonly employed assays are mentioned and briefly described. Apoptosis assays, based on methodology, can be classified into six major groups, and a subset of the available assays in each group is indicated and briefly discussed ... 

Examples best illnstrate the power of HCS. There is a tremendous variety of assays possible. This includes chemotaxis, morphological changes, nuclear translocation, snbcellnlar localization, cell-to-cell communication, cell viability, toxicity, micronuclei formation, cell cycle arrest, and receptor internalization. An NF-kB nuclear translocation assay, an apoptosis assay, and a gap jnnction screen are described below. Only the gap jnnction assay was performed at sanofi-aventis the other two examples were taken from an author s experience at Prelnx with the development of the precnrsor to the INCell 3000. 

Parameter of apoptosis Assay method Advantages Limitations... 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

Apoptosis is a genetically controlled 60 cell death. Various approaches are used in studying apoptosis, and to distinguish live cells from early and late apoptotic cells and from necrotic cells. Apoptosis assays may be based on ... 

Bogojevic D, Chamberlain MD, Barbulovic-Nad I, Wheeler AR (2012) A digital microfluidic method for multiplexed cell-based apoptosis assays. Lab Chip 12(3) 627-634... 

Figure 11.8 (a) Cell viability and (b) LDH leakage of Vero cells treated with p-G and f-G at different concentrations, (c) Flow cytogram showing apoptosis assay based on annexin V-FITC and PI staining of cells. Reproduced from ref. 142 with permission from the Royal Society of Chemistry. 

Major Applications Shelf life indicator, determining proteins, human serum albumins, polymeric biguanides,5 examining renal and hepatic functions, apoptosis assay ... 

Batchelor, R. H. Zhou, M. Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation applications in cytotoxicity and apoptosis assays. Anal. Biochem. 2004, 329, 35-42. 

---