A reliable preparation of mono-dispersed chromosome suspensions for flow cytometry |
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Authors: | M Stoehr K J Hutter M Frank Kl Goerttler |
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Institution: | (1) Institute for Experimental Pathology, German Cancer Research Center Heidelberg, Im Neuenheimer Feld 280, D-6900 Heidelberg, Germany |
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Abstract: | Summary 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. |
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