Tuesday, June 21, 2016

Using Automated Imaging and Cellular Analysis to Increase Throughput and Objective Result Generation in Genotoxicity Assays


Genotoxicity refers to the potential of in vivo or ex vivo agents, such as cancer-causing cellular mechanisms, environmental compounds, or chemical molecules to induce damage or alterations to DNA, genes and chromosomes. Testing for potential inducers that cause, or treatments that can repair DNA damage has implications for a number of life science research areas, including oncology, environmental, and cosmetic research. Several in vitro genotoxicity tests have been developed, and among the more popularly performed tests are the Ames test which uses histidine negative Salmonella bacterial strains; the micronucleus test where treated or test cells are visually examined for the presence and frequency of micronuclei created due to toxicity, the single cell gel electrophoresis (SCGE) or comet assay where fragmented (damaged) and undamaged DNA create a comet-like configuration in a gel upon electrophoresis and staining; and the gamma H2AX (γ-H2AX) assay where nuclear foci created from antibody labeled phosphorylated histone 2AX are a biomarker for genotoxic exposure.

The desire to perform genotoxicity tests in a higher throughput format has increased as a direct relationship to the incorporation of some of these tests to develop personalized medicines or to move away from the use of animal models. This is witnessed by the growing number of companies offering standardized "off-the-shelf" plates, kits, and reagents to perform these assays. In addition, contract research organizations (CROs) are now offering their services in this area to increase throughput.

While methods to improve throughput have advanced recently, the means to perform these higher throughput assays has in many ways stagnated. Imaging is still performed manually in many situations. Analysis can also be a daunting task, particularly in the case of the comet assay where manual hunting for the “correct” comet is performed, images are taken, and the process is then repeated…comet after comet, and well after well. Not only is this process extremely time consuming, but also builds in a great deal of subjectivity into the analysis. BioTek's Cytation™ 5 and Lionheart™ FX automated imaging systems and Gen5™ Microplate Reader and Imager Software can provide easy-to-use solutions to both automate the image capture process and create objective analyses of genotoxicity assays.

The first example shown below illustrates imaging and analysis of the comet assay. Following fluorescent staining, the Cytation 5 Cell Imaging Multi-Mode Reader was used to automatically image the samples. Unlike manual methods, simple and advanced inclusion/exclusion criteria were programmed into the Gen5 Microplate Reader and Imager Software to automatically place object masks around each separate comet head and tail to eliminate anomalies and other false objects that might be mistakenly included in manual analyses (Figure 1). The total fluorescence intensity in the tail portion of the comet, in relation to the total comet fluorescence, is used to calculate “Percent DNA in the Tail”. These calculations can then be combined with object size data to calculate the “Comet Tail Moment”.
Automated comet analyses to determine (A.) comet head and (B.) comet tail in relation to the comet head.
Figure 1. Automated comet analyses to determine (A.) comet head and (B.) comet tail in relation to the comet head.
The second example illustrates a nuclear stain imaging and analysis of the γ-H2AX assay. Immunofluorescence using a primary antibody specific to γ-H2AX and a Cy-5 labeled secondary antibody is initially performed followed by automated imaging. Two separate cellular analyses are then carried out using Gen5 software. The first to determined the average area of a single labeled phosphorylation site, or foci. The second to place object masks around individual nuclei using primary analysis parameters, and then secondary parameters to place masks around individual foci within each nuclei (Figure 2). The combined analyses allow for a number of metrics to measure DNA damage to be generated, such as labeled foci coverage area and calculated foci number per nuclei.
Automated γ-H2AX analyses to determine (A.) cellular nuclei per image and (B.) labeled foci within each nuclei.
Figure 2. Automated γ-H2AX analyses to determine (A.) cellular nuclei per image and (B.) labeled foci within each nuclei.
By incorporating automated imaging and analysis many of the headaches and limitations of performing in vitro genotoxicity assays can be eliminated. Countless hours do not have to be spent in front of a manual imager and objectively generated results have greater comparability from experiment to experiment. We encourage you to contact BioTek to find out more about how these assays can be performed.

By: BioTek Instruments, Brad Larson, Principal Scientist

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