In Vitro Cytokinesis-Block Micronucleus Assay | PraxiLabs

In Vitro Cytokinesis-Block Micronucleus Assay Virtual Lab Simulation

Biology | Toxicology | Biochemistry | Proteomics | Pharmacology



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General Aim

This experiment aims at detecting the damage of chromosomes, both chromosome loss, and chromosome breakage, to evaluate the induction of genotoxic effects of nanoparticles and nanomaterials using the light microscope.

Method

In Vitro Cytokinesis-Block Micronucleus Assay using Microscopy

Learning Objectives (ILO’s)

  • Successfully handle the required instruments and consumables needed in the experiment.

  • Check the confluence and count cells under the microscope.

  • Dilute the cells to a specific count suitable for seeding in the 24-well plate.

  • Calculate the concentration of tested chemicals and prepare the calculated doses in the cell culture medium.

  • Treat cells with the genotoxic agent(s) or nanoparticles and observe under the microscope Harvest cells, fix them and stain with Giemsa stain.

  • Analyze cells by light microscope and evaluate analyzed data.

  • Represent and interpret the resulting data graphically using dot plots.

Theoretical Background/Context

In genetics, genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

Principle of Work

The cytokinesis-block micronucleus assay (CBMN) is a sensitive and simple indicator of chromosome damage, both chromosome loss and chromosome breakage, which also provides information on cell cycle progression and cytotoxicity. MN are small nuclei separated from the main nucleus and contain chromosomes or chromosome fragments, derived from mitotic spindle dysfunction or acentric fragments. Because they are expressed in cells that have completed nuclear division, they are ideally scored in the binucleated stage of the cell cycle. 

 

 

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