In Vitro Micronucleus Assay
In Vitro Micronucleus Assay
Micronucleus formation is a hallmark of genotoxicity, and the micronucleus assay is an important component of genotoxicity screening. Micronuclei are chromatin-containing bodies that represent fragments or even whole chromosomes that were not incorporated into a daughter cell nucleus at mitosis. The purpose of the assay is to detect those agents that induce chromosome damage leading to the induction of micronuclei in interphase cells.
Micronuclei induction can result from clastogens (agents that induce chromosomal breaks mainly through interaction with the DNA) or aneugens (agents that induce chromosomal loss mainly through interference with the spindle apparatus). The major mechanisms responsible for micronucleus induction are double-stranded DNA breaks leading to micronuclei with acentric fragments (chromosomal fragments lacking a centromere). The complexity of the mitotic process means that alterations can also result in the failure of the chromosomes to segregate properly. Agents that interfere with the mitotic spindle apparatus (i.e. by affecting tubulin polymerization or spindle microtubule stability) can also induce micronuclei. These micronuclei are whole chromosomes that were unable to migrate with the rest of the chromosomes during anaphase.
The in vitro micronucleus assay is part of the recommended regulatory test battery for genotoxicity. It is an alternative to the in vitro chromosomal aberration assay. Multiple published studies show a high level of concordance (80-90%) between the in vitro micronucleus assay and in vitro chromosomal aberration assay. In addition, the in vitro micronucleus assay has the advantage that it is simpler, rapid, and it has more statistical power since it scores more cells; it can also detect aneuploid-inducing agents, which are very difficult to detect with the in vitro chromosomal aberration assay.
The automated in vitro micronucleus assay is conducted in a very similar way to the standard manual in vitro micronucleus assay, with the main difference being the scoring of the cells. The manual in vitro micronucleus assay uses trained operators to visually read slides under a microscope, whereas the automated assay uses proprietary image-analysis software to score the cells. Detecting micronuclei in binucleated cells is a straightforward process that makes this assay an excellent candidate for image-analysis based automation.
Eurofins In Vitro Micronucleus Assay
The in vitro micronucleus assay is conducted in CHO-K1 (Chinese Hamster Ovarian) cells, which are widely used for this assay due to their stable and well-characterized karyotype. The cells are seeded in 96-well plates and treated with the test compounds for 24 h (without S9) and for 4 h (with S9). Cytochalasin B, which is a cytokinesis blocker, is added after 24 h and the cells are incubated for an additional 24 h, after which the cells are fixed and scored for micronuclei. The assay is typically run using 8 concentrations in duplicate, and data for the highest 5 concentrations that are not cytotoxic are reported. Typically, 2,000 binucleated cells are scored per concentration, or 1,000 cells per well with n=2 wells. For compounds showing very high toxicity, fewer than 2,000 cells might be scored for the toxic concentrations. Duplicate wells that exhibit > 80% (n=2) cytotoxicity are noted as CYTO and are not scored.
After the experimental protocol is completed, the plates are scanned with an automated fluorescent microscope. The cellular nuclei and micronuclei are stained with Hoechst and the cellular cytoplasmic area is defined with a cell tracker fluorescent dye. The micronuclei are identified and counted in bi-nucleated CHO-K1 cells. A valid micronucleus should be located in the cytoplasmic cellular area, detached and of similar intensity to the main nuclei, and with a diameter less than 33% of the main nuclei.