Frequently Asked Questions
Q: How hard is it to learn to do the CometChip® assay?
A: Most researchers are able to generate publishable data within one or two experiments. The one step that benefits from practice is cell loading. CellArray provides a free CometChip® specifically to allow users to practice cell loading and to compare efficacy for two different well sizes (30 and 40 micron wells).
Q: How much faster is the CometChip® compared to the traditional comet assay?
A: Estimates vary because it depends on how many samples are being analyzed, but at a minimum, the method is 100X faster. What is more important is that it is possible to handle hundreds of samples at once, which is not doable when handling glass slides. Dr. Guo from the National Center for Toxicological Research led a project that involved analysis of over 2,000 conditions and over 200,000 comets, which would not have been possible using traditional methods.
Q: Is the CometChip® valuable even if I only want to test a few samples?
A: Yes. Even if you don’t use all 96 wells, your experiments will be more dependable and the assay is far easier to execute. The associated software enables automated scoring which greatly accelerates experiments.
Q: What classes of DNA damage can be detected using CometChip?
A: The CometChip® can be used to detect single strand breaks, double strand breaks, damaged bases, bulky lesions (including intrastrand crosslink), and interstrand crosslinks. Single strand breaks are readily detected using the alkaline comet assay conditions and double strand breaks are detectable using the neutral comet assay. To detect base damage, purified glycosylases can be added post lysis. Increase in signal with the glycosylase present indicates the extent to which that glycosylase’s substrates were present. To detect crosslinks, inhibition of DNA migration is used as a metric. For bulky lesions, HU AraC can be incorporated into the assay to trap nucleotide excision repair (NER) intermediates so that signal in the presence of HU/AraC is indicative of the presence of NER substrates (bulky lesions).
Q: How do I know which well size to use?
A: Both 30 micron and 40 micron well sizes are generally effective for most cell types. The 40 micron well size has been shown to be effective for WBCs, TK6, HepaRG, and HepG2 cell lines as well as HepRG cells and primary cells from liver, lung, kidney, pancreas, brain and spleen. The 30 micron well size has been shown to be effective for HepaRG cells. Of note, the background level of signal can go up if cells are very small (e.g., spleen cells), in which case the 30 micron well size is recommended.
Q: Can the CometChip® be used for any cell type? Are there cell types that don’t work well?
A: CometChip® accommodates virtually any cell type that has a spherical shape. Buccal cells do not load well into the microwells due to their flat shape. Neurons with projections are not anticipated to load well, however trypsinized neurons are spherical and load effectively into the CometChip wells. As described above, CometChip® has been shown to be effective for WBCs, TK6, HepaRG, and HepG2 cell lines as well as HepRG cells and primary cells from liver, lung, kidney, pancreas, brain and spleen.
Q: Is it possible to do both the alkaline and the neutral comet assay using CometChip®?
A: Yes. While most researchers use the alkaline comet assay conditions, the CometChip® can also be performed under neutral conditions to better reveal the presence of double strand breaks. S. Divkar et al. in the Roy laboratory used the CometChip to assess DSB repair in human lymphocytes. P. Sykora et al. in the Sobol laboratory measured DSBs in WBCs from turtles. Weingeist et al. and Tay et al. showed that double strand break repair kinetics can be revealed using the neutral CometChip® conditions.
Q: Is it possible to use CometChip® to do the “in vivo comet assay” wherein tissues or primary cells are analyzed to reveal the DNA damage levels that had been present in vivo?
A: Yes. Two laboratories have demonstrated that CometChip is effective for the “in vivo comet” assay. In the Azqueta laboratory, M. Collia et al. demonstrated that the CometChip yields consistent results with the two-well traditional comet assay. Of note, M. Collia et al. successfully performed the CometChip® sample processing on ice to reduce background damage levels. Additionally, N. Owiti et al. laboratory showed that the CometChip® is effective for assessing DNA damage in mice exposed to methylating agents. The method was effective for liver, lung, kidney, pancreas, brain, and spleen.
Q: Can the FLARE Assay (a.k.a. Enzyme-Reveal Assay) be performed on the CometChip?
A: Yes, bifunctional DNA glycosylases that cleave the backbone at sites of damaged bases can be used to reveal the presence of their substrates. For monofunctional glycosylases, a subset of abasic sites are cleaved under alkaline comet conditions, making it possible to reveal the presence of glycosylase substrates. It is recommended that the percent agarose for the overlay be reduced to 0.6% and the wash time in enzyme buffer should be increased to 3 x 15 min. It may also be advantageous to reduce the enzyme units per sample. Please see M. Collia et al.
Q: Can organoids be analyzed on the CometChip®?
A: While there is limited research in this space, C. Chao et al. showed that hepatocyte spheroids can be grown on-chip and analyzed when intact, thus preserving more normal cell behavior.
Q: Is it a problem if I have more than one cell per microwell?
A: Having several cells per microwell does not interfere with performing the CometChip® assay. There are dozens of publications wherein the experiments were performed with multiple cells per microwell. It is advisable to use conditions that are not significantly toxic to prevent apoptotic tails.