An improved lymphocyte culture technique: deoxycytidine release of a thymidine block and use of a constant humidity chamber for slide making.

The use of a thymidine block' for the induction of synchronous cell division in lymphocyte cultures is gaining in popularity in clinical cytogenetics. The excess thymidine, converted to thymidine triphosphate within the cell, inhibits the ribonucleotide reductase catalysed reduction of cytidine diphosphate to deoxycytidine diphosphate. This leads to a depleted deoxycytidine 5'-triphosphate pool which severely limits DNA synthesis.4A disadvantage of this technique is that release of the block requires the removal of the thymidine rich medium and its replacement with normal medium. This is a labour intensive and delicate operation subject to several sources of variability which can adversely affect the mitotic index and the chromosome length. However, it has been shown in cell lines derived from rodents,2 3 human leukaemic T lymphocytes.4 and Chang appendix cells' that a thymidine block can be released by the addition of 2-deoxycytidine to the thymidine rich culture medium. We have found that this approach can be successfully applied to 48 and 72 hour human lymphocyte cultures. A further significant cause of inconsistency in chromosome preparations is considered to be the atmospheric conditions, particularly the relative humidity, during slide making." We have eliminated this component of variability by making use of a waterbath as a constant humidity and temperature chamber. We therefore present a complete protocol for the production of good quality metaphase and prometaphase chromosome preparations from lymphocyte cultures, which differs from published methods in the use of 2-deoxycytidine to release a thymidine block with a constant humidity chamber for slide making.