Premature Chromosome Condensation in Human Resting Peripheral Blood Lymphocytes Without Mitogen Stimulation for Chromosome Aberration Analysis Using Specific Whole Chromosome DNA Hybridization Probes

Abstract

We have previously described a unique, simple, and rapid method for inducing premature chromosome condensation (PCC) in “resting” human peripheral blood lymphocytes (HPBLs) without mitogen stimulation and an approach for studying numerical changes and/or structural aberrations involving a specific pair of human chromosomes. The current protocol incorporates improvements that provide better PCC, incorporates a high-throughput automated sample preparation unit and metaphase harvester to minimize manual labor and improve quality, and supports simultaneous painting of multiple sets of human autosomes in interphase nuclei. To induce PCC, isolated HPBLs are incubated at 37 °C in cell culture medium supplemented with a phosphatase inhibitor (okadaic acid or calyculin A), adenosine triphosphate, and p34^cdc2/cyclin B kinase (an essential component of mitosis-promoting factor) for a short period of time. PCC spreads are prepared on glass slides using a humidity- and temperature-controlled chamber (an auto-spreader) after a brief hypotonic treatment and fixation. Aberrations involving specific sets of painted human chromosome are analyzed using fluorescence microscopy. Each of the normal (undamaged) painted homologous chromosome pairs displays two fluorescent spots, whereas cells with numerical and/or structural aberration involving specific painted chromosome sets show deviation in the number of fluorescent spots. The identification and quantification of aberration involving specific chromosomes in interphase nuclei have important applications in radiobiology, toxicology, radiation therapeutics, and cancer research.