Fluorescent G- and C-bands in mammalian chromosomes by using early Brd U incorporation simultaneous to methotrexate treatment

Fluorescent G- and C-bands were obtained in human and cattle chromosomes of lymphocytes grown at 37.5D̂ for 72–76 hours. 24 hours before the completion of the culture, 5-bromodeoxyuridine (BrdU) in a final concentration of 20 μg/ml and increasing doses of methotrexate (MTX) were added. After 17 hours the cells were washed and allowed to recover for 6 hours in a medium containing thymidine. Colcemid treatment lasted 1.5 hours. The air dried slides were stained with acridine orange and observed under fluorescence microscopy. Compared to the control (without MTX), it was possible by increasing the MTX doses to increase the number of cells in the first cycle of replication in the presence of BrdU (G-bands in both chromatids) with a concomitant reduction of the number of cells in the second cycle of replication (G-bands in one chromatid), which also allows demonstration of SCEs. The advantages of this technique and the different cellular responses between the two species are discussed.