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Wet Lab Radiation Damage Measured by Micronucleus (MN) Assay

Wet Lab Radiation Damage Measured by Micronucleus (MN) Assay. Background Equipment Supplies Procedures Lab Demonstrations. Background. Types of micronucleus assays Applications of micronucleus assays. MN. Background. Micronucleus Assay (MN).

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Wet Lab Radiation Damage Measured by Micronucleus (MN) Assay

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  1. Wet Lab Radiation Damage Measured by Micronucleus (MN) Assay • Background • Equipment • Supplies • Procedures • Lab Demonstrations

  2. Background • Types of micronucleus assays • Applications of micronucleus assays

  3. MN Background

  4. Micronucleus Assay (MN) • It can be used to detect the clastogenic and aneugenic effects of test agents both in vitro and in vivo. • Clastogenic: any substance or process causing chromosome breaks. • Aneugenic: Agents which affect cell division and the mitotic spindle apparatus resulting in the loss or gain of whole chromosomes, thereby inducing an aneuploidy. Background

  5. Micronucleus Assay (MN) • It is much more cost effective than the metaphase chromosome aberration assay. • The in vitro micronucleus assay is considered as the replacement for conventional metaphase analysis as the screening test of choice for clastogenicity. Background

  6. Micronucleus Assay (MN) • The in vivo assay, usually conducted in mice, is especially important since no in vitro alternative test has been validated to replace the MN test. • The cells evaluated in this assay are typically erythrocyte populations in either the peripheral blood or bone marrow compartment. Background

  7. Micronucleus Assays (MN) • Cytokinesis-block micronucleus (CBMN) assay • lymph, cell lines • Flow cytometric micronucleus (FCMN) assay • red blood cells Background

  8. CBMN Assay (lymphocytes) • Cytochalasin-B (actin inhibitor) Background

  9. Background

  10. CBMN + FISH Background

  11. Criteria for CBMN • The diameter of the MN should be less than one-third of the main nucleus. • MN should have similar staining as the main nucleus. • MN should be separated from or marginally overlap with main nucleus. Background

  12. Background

  13. CBMN vs. Chromosome Spreading • Advantages • Rapidity • Cheap • Simplicity • Statistical power • Disadvantages • Not all chromosome aberrations (acentric fragments) • Toxicity of cyto-B • Lymph. vs. cell lines Background

  14. Applications of MN Assay • Biological dosimetry • Risk assessment of cancer • Lung: smoking • Oral mucosa and bladder cancer: arsenic • Genotoxic effects of pesticides • Cytotoxic effects of ROS • Radiosensitivity indicator in head and neck tumor patients Background

  15. Dose Response MN chromosome 2.0 4.0 ??? % MN 1.0 2.0 Dicentrics/Cell Post-irradiation time 0 0 2 4 Dose (Gy) 1 2 Dose (Gy) Background

  16. FCMN Assay • Rapid scoring • Large number of cells • Small changes in MN frequency • Reticulocytes from peripheral blood • DNA contents

  17. Where are micro-nucleated cells? DNA content

  18. Whole blood X White blood cells Red blood cells (size) X X Platelets (anti-platelet) CD61 Reticulocytes (CD71) Nucleated red cells (DNA contents) Litron Laboratory Steve Dertinger, Ph.D. Ret-MN

  19. Cell size (red cells)

  20. Single cells total mature RBC Single cells platelets (anti-CD61-PE) Single cells Reticulocytes for MN

  21. MN Single cells Reticulocytes No platelets MN: DNA content

  22. Equipment • Tissue culture equipment • Sterile tissue culture hood, Incubator (CO2, 37oC, humidified), Microscopes( upright, inverted, dissecting), Pipettes, Hemacytometers (Coulter counter). • Radiation source • Flow cytometer

  23. Supplies • Tissue culture flasks (dishes) • Tissue culture medium • Glass slides • Cytochalasin-B • Giemsa stain • MN DNA staining kit (Litron)

  24. ProceduresCalibration and color compensation for flow cytometer • Rat blood cells • DNA (PI), CD71- (FITC), anti-platelets+ (PE) • Parasite infected mouse blood cells • DNA (PI), CD71+ (FITC) • Parasite infected mouse blood cells • DNA (PI), CD71+ (FITC), anti-platelets+ (PE)

  25. Single cells mature RBC Rat blood cells DNA (PI) CD71- anti-platelet Single cells platelets (anti-CD61-PE) Single cells Reticulocytes for MN

  26. Rat blood cells

  27. Parasite infected mouse blood cells DNA (PI) CD71+ DNA ratio 20:6000 (anti-CD61-PE)

  28. Parasite infected mouse blood cells

  29. Parasite infected mouse blood cells DNA (PI) CD71 anti-platelet (anti-CD61-PE)

  30. Parasite infected mouse blood cells

  31. MN Unirradiated (0 Gy) mouse blood cells DNA (PI) CD71+ CD61+

  32. Parasite infected mouse blood cells by FACS Vantage sorter DNA (PI) CD71+ CD61+

  33. Human blood cells from irradiated (therapy) patient DNA (PI) CD71+ CD61+

  34. Lab Demonstrationsroom 3-4151, 3-4157 • FCMN assay room B-6624, B-6625 • CBMN assay

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