COMPAC-50 is a high throughput electrophoresis system, designed to perform the COMET Assay, otherwise known as Single Cell Gel Electrophoresis. A unique patent pending design employs two carriers to hold a total of 50 slides (25 per carrier) vertically. This provides two distinct advantages over conventional Comet Assay systems that utilise a horizontal platform for manual mounting of multiple individual slides. Firstly to produce a highly compact system which saves 75% of Lab space. Secondly by holding 25 slides in a rack this allows slides to be processed together in one batch saving on handling assay time by up to 90%. Consequently, this is not only beneficial for electrophoresis but also in the lysis, neutralisation, staining and washing steps of the Comet Assay, when each batch of slides may be treated during each step respectively using the four ebony acrylic staining dishes supplied. In addition, the COMPAC-50 benefits from a high performance ceramic cooling base with sliding drawer to accommodate a cool pack, which is frozen before use, to maintain optimal buffer temperature.
COMPAC-50 – Experimental Procedure
Overview of a typical comet assay procedure. A single-cell suspension of the cells under investigation is mixed with low melting point agarose. The cell/agarose mix is layered on glass microscope slides, pre-coated with agarose, and the agarose allowed to set. The cells are lysed under high pH before washing with pure water. The presence of strand breaks and high pH allows the cellular DNA to unwind. Electrophoresis draws the DNA out of the nucleoid body forming a ‘tail’. The amount of migration (the amount
of DNA in the tail versus the head) is proportional to the initial amount of DNA damage. The slides are then drained, neutralised and washed with pure water before drying overnight. Following further washing in pure water the slides are stained, washed and finally scored and analysed, typically using fluorescent microscopy and image analysis software. Advantageously, the use of a slide-holder assembly, which maintains multiple slides in laminar arrangement, also permits batch processing of these slides through pre-electrophoresis and post electrophoresis steps, thus eliminating the need for individual manipulation of samples.
Background to COMET Assay: First introduced in 1981 to quantify double-stranded DNA breakages in single cells exposed to g-irradiation, the Comet Assay (or SCGE) has since been adapted to analyse specific DNA lesions and repair processes.
Overview: Following genotoxic insult, such as ionizing radiation, the resultant strand breakage of supercoiled duplex DNA reduces the size of the large genomic DNA from which these strands are separated or drawn out by electrophoresis. The genomic DNA then takes on the appearance of a ‘comet’ as its negatively charged broken ends and fragments migrate towards the anode during electrophoresis.
Method: After exposure to a genotoxic insult cells are suspended within low melting point agarose and embedded within a thin layer of agarose on a microscope slide. Cellular protein is then removed by lysis in detergent, when DNA is allowed to unwind in alkaline conditions before electrophoresis. The DNA is electrophoresed, stained and then analysed using software.
Results: Microscopy imaging is used to measure DNA fluorescence upon staining. In DNA damaged cells the resultant image resembles a ‘comet’ with the cellular DNA separated into a head and tail. The head is mainly composed of intact genomic DNA, whereas any fragmented or damaged DNA is concentrated within and towards the tail.
*developed in collaboration with the Oxidative Stress Group in the Department of Cancer Studies and Molecular Medicine within the University of Leicester.