Wet Lab

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Wet Lab Radiation Damage Measured by
Micronucleus (MN) Assay

• Background
• Equipment
• Supplies
• Procedures
• Lab Demonstrations

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Background

• Types of micronucleus assays
• Applications of micronucleus assays

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MN
Background
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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
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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
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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
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Micronucleus Assays (MN)

• Cytokinesis-block micronucleus (CBMN) assay
• lymph, cell lines
• Flow cytometric micronucleus (FCMN) assay
• red blood cells

Background
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CBMN Assay (lymphocytes)

• Cytochalasin-B (actin inhibitor)

Background
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Background
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CBMN FISH
Background
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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
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Background
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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
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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
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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
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FCMN Assay

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

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Where are micro-nucleated cells?
DNA content
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Whole blood
X
White blood cells
Red blood cells (size)
X
X
Reticulocytes (CD71)
Platelets (anti-platelet) CD61
Nucleated red cells (DNA contents)
Litron Laboratory Steve Dertinger, Ph.D.
Ret-MN
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Cell size (red cells)
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Single cells total mature RBC
Single cells platelets
(anti-CD61-PE)
Single cells Reticulocytes for MN
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MN
Single cells Reticulocytes No platelets
MN DNA content
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Equipment

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

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Supplies

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

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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)

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Single cells mature RBC
Rat blood cells
DNA (PI) CD71- anti-platelet
Single cells platelets
(anti-CD61-PE)
Single cells Reticulocytes for MN
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Rat blood cells
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Parasite infected mouse blood cells
DNA (PI) CD71
DNA ratio 206000
(anti-CD61-PE)
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Parasite infected mouse blood cells
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Parasite infected mouse blood cells
DNA (PI) CD71 anti-platelet
(anti-CD61-PE)
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Parasite infected mouse blood cells
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MN
Unirradiated (0 Gy) mouse blood cells
DNA (PI) CD71 CD61
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Parasite infected mouse blood cells by FACS
Vantage sorter
DNA (PI) CD71 CD61
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Human blood cells from irradiated (therapy)
patient
DNA (PI) CD71 CD61
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Lab Demonstrationsroom 3-4151, 3-4157

• FCMN assay

room B-6624, B-6625

• CBMN assay