Methotrexate-leucovorin synchronization of human lymphocyte cultures. Induction of high resolution R- and G-banding.

This report describes simple techniques for induction of high resolution R- and G-banding. A methotrexate (MTX) induced S-phase block was released after 18 h by exposure to leucovorin (LV). G-bands were induced by adding ethidium bromide (EB) to the cultures followed by a modified trypsin treatment of the slides, including inhibition of trypsin activity by trypsin inhibitor. To obtain R-banded preparations 5-bromodeoxyuridine (BUdR) and Hoechst 33258 (H) were simultaneously added to the LV released cultures. The synchronization technique and the R- and G-banding procedures are simpler than most techniques, formerly published, and ensure high metaphase yield, reproducibility and slides with well defined high resolution R- or G-banding.