Abstract

The Science that Motivates this Protocol: Environmental and endogenous sources of DNA damage promote cancer, aging and other diseases. Ironically, at much higher doses, clinicians use DNA damaging agents to treat cancer. A complex network of DNA damage responses modulate the risk of cancer and disease, and that also modulate the efficacy of cancer chemotherapeutics and radiotherapy. Despite the impact of DNA damage and repair on health, few methods are available for high throughput assessment of DNA damage and DNA repair kinetics in human cells.

The Approach: To overcome this limitation, Wood et al. (referenced below) developed a method to create a cell array in agarose. DNA damage is analyzed using methods that are similar to the traditional comet assay, which is effective for detection of single strand breaks, abasic sites, alkali sensitive sites, base lesions that can be enzymatically cleaved, double strand breaks, and crosslinks. DNA damage levels are measured based upon the underlying principle that damaged DNA migrates more readily than undamaged DNA when electrophoresed. The approach exploits a cell microarray that is created by trapping cells into microwells that can be as small as the diameter of a single cell. The microwells are in agarose, which makes it possible to culture, challenge, and measure DNA damage levels in almost any cell type. Cells can repair their DNA in the array, enabling studies of DNA repair kinetics. Arranging cells on a single plane reduces the number of images required. The array suppresses comet to comet interference. Using this approach, compared to the traditional comet assay, throughput is increased by about two orders of magnitude, and sample-to-sample variation is suppressed.

The NextGen Protocol: This protocol describes how to load cells into the matrix, assemble the hardware, and process the sample. Key details regarding approaches for getting a clean cell array (without spurious cells) are described, as well as optional conditions that can be used to tune the loading conditions for a particular cell type. The platform incorporates standard 24- or 96-well formats, which are compatible with other high-throughput screening (HTS) tools.

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