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Microbeam Training Course RARAF 2011

Microbeam Training Course RARAF 2011. MICROBEAM LABORATORY 3 Sample Image Capturing & Analysis RARAF Experiences – Micronuclei and DNA damage foci formation Identifying micronuclei Charles Geard & Brian Ponnaiya.

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Microbeam Training Course RARAF 2011

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  1. Microbeam Training Course RARAF 2011 MICROBEAM LABORATORY 3 Sample Image Capturing & Analysis • RARAF Experiences – Micronuclei and DNA damage foci formation • Identifying micronuclei Charles Geard & Brian Ponnaiya

  2. RARAF Experiences Micronuclei and DNA damage foci formation

  3. Human fibroblasts AG1522 G1 phase Microbeam

  4. Normal human keratinocytes: BrdU labeled & FITC immunofluorescence plus PI counterstain for non-cycling cells, 24 hr after microbeam nuclear irradiation Micronuclei per cell pair Micronucleus & bridge

  5. Nucleoplasmic bridges & micronuclei in BrdU tagged normal human melanocyte cell pairs [highly motile]

  6. Nucleoplasmic bridge extension in a labeled melanocyte pair 72 hr post microbeam particles to a nuclear centroid

  7. 2.5Gy, 24hr 2 2 6 13 2.5Gy, 24hr 2 RGB display RGB display Pseudocolor Pseudocolor Identification of chromosomes involved in micronuclei by M-FISH DNA ligaseIV -/- cells

  8. γ-H2AX TRF2 Merge 200 -particles t ~ 10 min 400 -particles t ~ 10 min Susan Bailey, CSU • TRF2 is not recruited to damage sites following exposure to high fluences of -particles

  9. γH2AX MDC1 Merge 400 -particles t = 30 min Results • Significant co-localization of DNA damage markers can be seen following localized -particle exposure • Later time points (30m and 60m) show an increase in the size of regions marked by γH2AX or MDC1

  10. Irradiated

  11. Bystander

  12. H33342 hit nuclei versus non-hit Cyto-orange tagged cells Keiji Suzuki, Nagasaki University Irradiated Bystander

  13. Microbeam irradiation of cell nuclei in a circle format for gamma H2A-X expression in hit and bystander AL cells TomooFunayama Japan Atomic Energy Research Institute

  14. 3.2 Gy 1GeV Fe 1h 30 min 3.2 Gy 1GeV Fe 24 hrs

  15. Criteria for identifying and scoring of micronuclei • Morphologically identical but smaller than nuclei (82%) • Round or oval in shape (99%) • Diameter between 1/3rd and 1/16th of main nuclei (91%) • Not linked to main nucleus (73%) • May overlap or touch main nucleus (49%) • Same color as nucleus (85%) • Similar staining intensity as nucleus (83%) Adapted from: Bonassi, et. al., Environmental and Molecular Mutagenesis 37:31-45 (2001) A comparison of micronuclei scoring in 25 laboratories in 16 countries

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