Apoptosis is is a process of programmed cell death that biochemical events lead to characteristic cell changes, including compaction of the nuclear chromatin, shrinkage of the cytoplasm and production of membrane-bound apoptotic bodies. As with cell viability, no single parameter fully defines cell death in all systems; therefore, it is often recommended to use several different methods when studying apoptosis. There are also various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow cytometry), cellular markers such as DNA fragmentation, caspase activation, Bid cleavage, and cytochrome c release (Western blotting) for analysis of apoptotic cells. Anti-cancer drug candidates failing to induce apoptosis are likely to have decreased clinical efficacy, making apoptosis assays important tools for high-throughput drug screening.
Last time, we discussed using highly fluorescent annexin V conjugates to detect apoptosis based on membrane phospholipid asymmetry changes during apoptosis. Here we decribe several assays using nucleic acid stains for detecting apoptotic cells.
Part1: Comet (Single-Cell Gel Electrophoresis) Assay to Detect Damaged DNA
The comet assay (single-cell gel electrophoresis) is a useful technique for studying DNA damage and repair with manifold applications. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. Cells are immobilized in a thin agarose matrix on slides, and then these embedded cells are then treated with a lysis buffer and alkaline solution, which relaxes and denatures the DNA. When subjected to electrophoresis, the unwound, relaxed DNA migrates out of the cells. Following electrophoresis, the samples are dried, stained with a DNA dye, and visualized by epifluorescence microscopy (Figure 1). Comet assays have traditionally been performed using ethidium bromide to stain the DNA; however, use of the SYBR Gold and SYBR Green I stains ref improves the sensitivity of this assay.
Figure 1. Scheme for the performance of the comet assay
After staining with a nucleic acid stain, cells that have accumulated DNA damage appear as fluorescent comets, with tails of DNA fragmentation or unwinding. In contrast, cells with normal, undamaged DNA appear as round dots, because their intact DNA does not migrate out of the cell (Figure 2).
Figure 2. Typical result of Comet Assay
The comet assay is an extremely sensitive DNA damage assay. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Essentially, anything that can cause DNA damage or denaturation except the factor(s) being researched is to be avoided. The most common form of the assay is the alkaline version, introduced by Singh and coworkers, detects a broad spectrum of DNA lesions, that is, DNA single- and double-strand breaks and alkalilabile sites. Due to its simple and inexpensive setup, it can be used in conditions where more complex assays are not available.
To be continued….