SIMPLIFIED AND OPTIMIZED KINETOCHORE DETECTION - CYTOGENETIC MARKER FOR LATE-G(2) CELLS

Cytogenetic detection of kinetochore proteins using the CREST antibody coupled with secondary antibodies labeled with different fluorescent probes has been optimized for several in vitro mammalian cell lines. T his study investigated selected parameters including the influence of common fixatives (methanol, ethanol, methanol:acetic acid (3:1)), dete rgents (Triton-X100, Tween), fluorescent probes (CY3, BODIPY, FITC), w ashing protocols, and an antifading agent (paraphenylenediamine) on th e detection of kinetochore proteins in control and X-ray (240 kVp)-irr adiated cells. Utilizing an optimized fixation and staining protocol, a brilliant visualization of kinetochores in interphase cells was obta ined in control as well as X-ray-irradiated interhase cells. Applicati on of this improved kinetochore staining methodology readily permits d iscriminating cells containing either single or paired kinetochores, t he latter of which are characteristic of late-G(2) phase and prophase cells.