Home › Cell Biology › CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
Cell Biology JoVE (Open Access) Citable · DOI

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DOI: 10.3791/50607-v
What you'll learn
  • ✓ Perform high-throughput DNA damage detection using CometChip microarray platform
  • ✓ Execute alkaline and neutral comet assays on 96 samples in parallel
  • ✓ Quantify DNA damage and repair kinetics in mammalian cells
Protocol

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

Difficulty
advanced
Total time
~4–6 hours per experimental run (including cell loading, dosing, assay incubation, and imaging)
Model organism
Human lymphoblastoid cells
Biosafety
BSL-1

Steps

1
Prepare CometChip device and substrate

Set up the microarray chip and apply necessary surface treatments or coatings to enable cell attachment and microarray patterning.

▶ 01:24
2
Load mammalian cells into CometChip array

Seed and pattern human cells into the 96-well microarray format on the CometChip device, ensuring uniform distribution across all wells.

▶ 02:20
3
Apply DNA-damaging dose and incubate repair

Expose cells to a controlled dose of DNA-damaging agent and allow repair time to proceed under specified conditions.

▶ 03:57
4
Perform alkaline comet assay protocol

Execute lysis, alkaline unwinding, and electrophoresis steps under conditions that detect single- and double-strand breaks.

▶ 04:55
5
Perform neutral comet assay protocol

Execute lysis, neutral unwinding, and electrophoresis steps to selectively detect double-strand breaks without single-strand break detection.

▶ 06:23
6
Image and acquire fluorescent comet data

Acquire high-resolution fluorescent images of stained DNA comets across all 96 wells using automated microscopy.

▶ 07:50
7
Quantify DNA damage and repair kinetics

Analyze comet images to extract DNA damage metrics (tail length, tail moment, % DNA in tail) and calculate repair rates from time-course data.

▶ 08:21
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