title: CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells.
abstract: DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The 'CometChip' is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology; jing_ge@mit.edu.
authors.forename: Jing
authors.initials: J
authors.lastname: Ge
authors.affiliation: Environmental Toxicology, Chulabhorn Graduate Institute.
authors.forename: Somsak
authors.initials: S
authors.lastname: Prasongtanakij
authors.affiliation: Department of Biomedical Engineering, University of Minnesota.
authors.forename: David K
authors.initials: DK
authors.lastname: Wood
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology.
authors.forename: David M
authors.initials: DM
authors.lastname: Weingeist
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology.
authors.forename: Jessica
authors.initials: J
authors.lastname: Fessler
authors.affiliation: Environmental Toxicology, Chulabhorn Graduate Institute.
authors.forename: Panida
authors.initials: P
authors.lastname: Navasummrit
authors.affiliation: Environmental Toxicology, Chulabhorn Graduate Institute.
authors.forename: Mathuros
authors.initials: M
authors.lastname: Ruchirawat
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology.
authors.forename: Bevin P
authors.initials: BP
authors.lastname: Engelward
mesh_terms: D020552:Comet Assay / Q000295:instrumentation* / Q000379:methods*; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004260:DNA Repair; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods; D006801:Humans; D055616:Microtechnology / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*
chemical_list: D004247:DNA