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Biochemistry JoVE (Open Access) Citable · DOI

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

DOI: 10.3791/52763-v
What you'll learn
  • ✓ Isolate leukocytes from blood samples for comet assay analysis
  • ✓ Perform lysis and electrophoresis to measure DNA damage
  • ✓ Interpret comet assay results as an indirect oxidative stress marker
  • ✓ Apply comet assay as a systemic oxidative stress assessment tool
Protocol

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Difficulty
intermediate
Total time
~3–4 hours per sample batch (including lysis, electrophoresis, and imaging)
Model organism
Human (blood-derived leukocytes)
Biosafety
BSL-1

Steps

1
Isolate leukocytes from whole blood

Extract and prepare leukocytes from blood samples using standard isolation techniques. This isolated cell population serves as the substrate for DNA damage assessment.

▶ 01:31
2
Perform cell lysis and electrophoresis

Lyse cells under controlled conditions and subject DNA to electrophoresis in an alkaline environment to separate and visualize DNA fragments. DNA breaks migrate differentially based on damage severity.

▶ 03:44
3
Analyze comet assay measures of oxidative stress

Quantify DNA damage patterns (comet tail length, intensity, and migration) using microscopy and image analysis software. These measurements correlate with the level of systemic oxidative stress present.

▶ 06:14
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