Home › Cell Biology › Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells
Cell Biology JoVE (Open Access) Citable · DOI

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

DOI: 10.3791/56265-v
What you'll learn
  • ✓ Apply laser micro-irradiation to induce localized DNA damage in mammalian cell nuclei
  • ✓ Monitor recruitment of fluorescent DNA repair proteins to damage sites using confocal microscopy
  • ✓ Distinguish single and double strand break repair mechanisms through live-cell imaging
Protocol

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Difficulty
advanced
Total time
~2–4 hours per experiment (including setup, irradiation, and image acquisition)
Model organism
Mammalian cultured cells (HEK293 or similar)
Biosafety
BSL-1

Steps

1
Set up laser micro-irradiation microscopy system

Configure confocal fluorescence microscope with laser micro-irradiation capability, calibrate laser intensity and focus, and prepare live-cell imaging chamber. Ensure proper wavelength selection for DNA damage induction.

▶ 00:31
2
Perform live-cell image analysis of protein recruitment

Acquire time-lapse confocal images of fluorescently-tagged DNA repair proteins localizing to micro-irradiated sub-nuclear regions. Track protein dynamics and accumulation at damage sites over time.

▶ 04:02
3
Characterize DNA damage and repair responses

Analyze image data to distinguish single and double strand break repair pathways, assess damage detection kinetics, and quantify repair protein signaling intensity and recruitment patterns.

▶ 05:54
💬 Comments coming soon