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by Olivier De Bardonneche last modified 14-06-2010 09:10

There is now a wide range of medium- to high-throughput comet assay formats.


•    Twelve-gel slides: designed for incubation of gels with different chemicals, enzymes or DNA hybridisation reagents, the 12-gel slides are used in combination with a chamber with a tight-fitting silicone (PDMS) gasket to      separate the gels. Two slightly different designs are available.  

 slide

•    GelBond films, accommodating 48 or 96 gels. The films are held taut in simple sprung wire frames, for ease of transfer between different solutions during the comet assay processing.
•    Modified Cell Array: slides with a ‘micropatterned’ surface, which allows cells to attach individually or in small groups. Adhered single cells are treated in parallel with a series of chemicals at different concentrations using a silicone mould. Agarose gel is then laid on the slide containing an array of about 100 cultures 
In the GelBond and 12-gel formats, each gel can contain up to a few hundred cells. The Cell Array system shows approximately 100 patterns per well and thus around 80 single cells (80% pattern filling).
Accessories:
•    Electrophoresis tank (‘White Cat’) designed to accommodate 4 GelBond films or 20 slides.
•    ‘Smart Cube’ electrophoresis tank holding a vertical stack of slides or larger plates.
•    Coated slides with superior binding properties

 

The cell array system as a parallel assay for cytotoxicity


The Cell Array system is designed for parallel testing of genotoxicity and cytotoxicity. Cells adhering to the micropatterned array show normal growth characteristics and metabolic activity (phase I enzymes tested in HepaRG cells). As a general cytotoxicity assay, the MitPT CJ1 test for loss of mitochondrial membrane potential has been successfully used in cell array conditions.


Optimal cell types for use in genotoxicity and cytotoxicity testing


Many different cell types can be used for genotoxicity testing in the comet assay – whether the standard assay or the new high throughput versions.
However, HepaRG cells, derived from liver, have unusually useful features as a model cell culture system, as they can be grown in an undifferentiated state to confluence, and then induced by treatment with DMSO to differentiate into hepatocytes or biliary cells. The hepatocytes, easily separated from the others, are applied to the Cell Array slides.  

Increased speed and efficiency of comet scoring


The Imstar Pathfinder instrument and dedicated Comet programme for comet assay image capture and analysis has been modified to scan patterned slides in the 12-, 48- and 96-gel formats, and specific algorithms developed to detect comets for a variety of cell types, with different morphology. This is a fully automated scoring system, essential when so many gels require scoring.
An alternative scoring method employs differential fluorescence of head and tail DNA. Head DNA – mainly double-stranded – is detected with a stain such as DAPI (or a novel stain characterised by TATAA) with high dsDNA specificity, while the predominantly ssDNA in the tail is hybridised with fluorescent-tagged total human genomic DNA probes labelled e.g. with biotin which is antibody-labelled with Texas red. This dual labelling approach is at the proof of principle stage.

Enhanced sensitivity of comet assay, by applying lesion-specific enzymes


Lesion-specific glycosylases/endonucleases are employed in the comet assay to detect oxidised and alkylated bases, UV-induced pyrimidine dimers, and misincorporated uracil. The glycosylase removes the base, leaving an AP-site (apurinic/apyrimidinic site). An associated AP lyase activity converts the AP-site to a break.
Current application of the comet assay in genotoxicity testing uses simple DNA breaks as the endpoint, with the result that some known genotoxins are not detected. The use of enzymes such as formamidopyrimidine DNA glycosylase (FPG) with the comet assay in genotoxicity testing increases sensitivity by detecting altered bases and AP-sites in addition to breaks. This approach promises to increase substantially the ability of the assay to detect genotoxic effects at low concentrations of suspect chemicals.

Development and comparison of methods for measuring DNA repair in vitro


‘Biochip’ assays have been developed for simultaneous assay of several repair activities in a cell or nuclear extract. The Oligo Biochip uses oligonucleotides with specific lesions fixed to a glass surface, and glycosylase/lyase/endonuclease activities (i.e. the early stages of repair) are detected by cleavage of the oligonucleotide, releasing a labelled fragment. The Plasmid Biochip detects repair synthesis on a plasmid containing lesions repaired by BER or NER pathways. These assays have been successfully applied to a variety of cultured cell types.
Comet-based DNA repair assays employ specifically damaged substrate cells, embedded in agarose and lysed, and incubated with cell extract; repair incision activity in the extract is measured as an accumulation of DNA breaks. BER and NER activities have been measured in human lymphocyte extracts; intra-individual variability is quite low, compared with the inter-individual range of 3-fold for BER and 7-fold for NER.

Detection of gene-specific DNA damage and repair using specific probes


Padlock probes are oligonucleotides designed to lock onto their hybridisation target sequences. Padlock probes for 3 genes – XPD, OGG1 and HPRT – have been tested for hybridisation to comet DNA. Rolling circle amplification and labelled oligonucleotide detection gives good signals, especially on comet tails containing single-stranded, hybridisable DNA. Preliminary experiments indicate fast repair of HPRT relative to total DNA.

Comet hybridised 

 Calibration of the comet assay


The results from the X-ray calibration trial show that (i) chemical dosimetry is useful for approximate comparisons of radiation doses; (ii) there is little repair of DNA strand breaks during processing of irradiated samples for comet analysis; (iii) the comet assay protocol applied to microgels on GelBond gives results very similar to those obtained with the traditional comet protocol; and (iv) scoring of comets with a semi-automated image analysis system gives somewhat higher % tail DNA levels than scoring with an automated system. The calibration trial identifies several factors that contribute to the different levels of DNA damage which have been reported for irradiated cells in the past.

Validation of the comet assay in its various forms


The 12-gel slide and 96-gel GelBond formats have been compared with standard slides carrying 2 or 3 gels, at several levels: (i) Damage was induced in lymphoblastoid cells by a range of concentrations of MMS, in 3 laboratories. Low levels of intra- and inter-laboratory variation were found. (ii) Three different scoring methods were compared, with MMS and H2O2-treated cells. Visual scoring, semi-automated and automated image analysis proved equally capable of detecting low levels of damage, and gave similar dose-response curves. (iii) Known genotoxic and control non-genotoxic chemicals were tested in 3 centres, using standard and new comet assay formats. In all cases, in terms of the results obtained,  the new and old methods are essentially interchangeable.
 
Reference and internal comet assay standards


Until now, reference standards in the comet assay have consisted of slides containing cells with a known amount of damage (negative and positive controls) run in parallel with the sample slides. A true internal standard (the ideal) requires standard reference cells that, after processing as comets, can still be distinguished from sample cells in the same gel. The approach that we have followed is to use cells of very low DNA content. Such cell samples could then be mixed and processed with sample cells, in the same gel, and separate cell type-specific analysis of DNA damage carried out, giving profiles of % tail DNA for both populations of comets, i.e. from sample cells and reference cells. Fish erythrocytes were tested for this role since (unlike mammalian cells) they contain nuclear DNA, and some species have several times less DNA than do human cells. Of several candidates tried, the turbot was demonstrated to be most suitable. A simple and cost-efficient alternative for reference cells has therefore been established, and procedures for the preparation and use of fish blood cells are available.

Commercialisation of novel products, equipment and materials


Products developed during the Comics project are the basis for our commercialisation plan. A ‘shopping basket’ approach will be used, so that customers can purchase just those components that they require, but comprehensive kits will also be available.

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