A reliable preparation of mono-dispersed chromosome suspensions for flow cytometry
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A reproducible, simple and rapid protocol was developed to prepare mono-dispersed chromosome suspensions for flow cytometric analysis. The procedure basically employes: 1. twice rinsing of monolayer cultures to eliminate dead cells and cellular debris, 2. mild fixation of mitotic cells with 1% acetic acid, and 3. ultra sonic treatment to release single metaphase chromosomes. The procedure takes less than 30 min. Isolated chromosomes are storable over months at 4° C. Despite mild fixation many biochemical studies and experiments applied on such fixed chromosomes purified and enriched using electronic sorting are feasible: 1. gene and restriction mapping, 2. cloning of specific gene sequences, and 3. gene frequency analysis.
KeywordsAcetic Acid Dead Cell Frequency Analysis Flow Cytometric Analysis Gene Frequency
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- Carrano AV, Gray JW, Langlois RG, Burkhardt-Schultz KJ, Van Dilla MA (1979) Measurement and purification of human chromosomes by flow cytometry and sorting. Proc Natl Acad Sci USA 76:1382–1384Google Scholar
- Cram LS, Arndt-Jovin DJ, Grimwade BG, Jovin TM (1979) Fluorescence polarization and pulse width analysis of Chromosomes by a flow system. J Histochem Cytochem 27:445–453Google Scholar
- Gray JM, Carrano AV, Steinmetz LL, Van Dilla MA, Moore DH, Mayall BH, Mendelsohn ML (1975) Chromosome measurement and sorting by flow systems. Proc Natl Acad Sci USA 72:1231–1234Google Scholar
- Gray JM, Peters D, Merrill JT, Martin R, Van Dilla MA (1979) Slit-scan flow cytometry of mammalian chromosomes. J Histochem Cytochem 27:441–444Google Scholar
- Otto F, Oldiges H (1978) Requirements and procedures for chromosomal DNA measurements for rapid karyotype analysis in mammalian cells. In: Lutz D (ed) Third international symposium on pulse cytophotometry. European Press, Ghent, Belgium, pp 393–400Google Scholar
- Sillar R, Young BD (1981) A new method for the preparation of metaphase chromosomes for flow analysis. J Histochem Cytochem 29:74–78Google Scholar
- Stöhr M, Goerttler KL (1979) The Heidelberg flow analyzer and sorter (HEIFAS) approach on the prescreening of uterine cancer. J Histochem Cytochem 27:564–566Google Scholar
- Stöhr M, Hutter KJ, Frank M, Futterman G, Goerttler KL (1980) A flow cytometric study of chromosomes from Rat Kangaroo and Chinese hamster cells. Histochemistry 67:179–190Google Scholar
- Van Dilla MA, Carrano AV, Gray JM (1976) Flow Karyotyping: Current status and potential development. In: Medelsohn ML (ed) Automation of cytogenetics U.S. Energy Research and Development Administration CONG-751158, pp 145–164Google Scholar