Immunofluorescent staining of kinetochores in micronucleus for detection of aneuploidy inducing agents.
Channarayappa; Nath-J; Ong-T
J Tissue Cult Methods 1992 Sep; 14(3):125-132
A simple and reproducible technique was developed for the differentiation of clastogens from aneuploidogens using immunofluorescent staining of kinetochores. In the immunofluorescent staining technique, cultured V79 cells were prepared on glass slides which were subsequently chemically treated, fixed in-situ and stained. The primary antibody used was the antikinetochore antibody with fluoresceinated goat antihuman immunoglobulin-G (IgG) as a secondary antibody to locate kinetochores. To identify the nucleus, micronucleus, and cytoplasm, 4,6-diamidino-2-phenyl-indole (DAPI) or propidium-iodide was used. Kinetochores appeared yellow/green against the orange/red nuclear material. The cytoplasm appeared faint green in slides counter stained with propidium-iodide. Kinetochores appeared apple green and the nucleus and micronucleus appeared light blue in DAPI counter stained slides. This indicated that the location of kinetochores and nuclear material within the cell boundary is achievable. The proportion of kinetochore positive and kinetochore negative micronuclei can be useful in determining the clastogenic and aneuploidogenic properties. This simple method can be applied to a variety of cells.
NIOSH-Author; Analytical-methods; Genotoxic-effects; Cytology; In-vitro-studies; Mammalian-cells; Cell-cultures; Chromosome-damage
Journal of Tissue Culture Methods