DNA extraction during Giemsa differentiation of chromatids singly and doubly substituted with BrdU

Abstract

Human lymphocytes were cultured in 3H-labelled BrdU. Cells were pretreated to induce differentiation, autoradiographed and Giemsa-stained. DNA extraction was deduced if grain counts were lower in differentiated mitoses compared with untreated controls.--The differentiation method involved sequential pretreatments with short wave UV and 2 x SSc at 60 degrees C. This removed 34% of label from first division cells (with TB.TB chromosomes) but relatively more (53%) from second division (TB.BB chromosomes). In second division cells, about two thirds of label was lost from pale (BB) chromatids but only one third from dark (TB) chromatids. The UV and SSC pretreatments acted in collaboration, since neither alone reduced grain counts significantly.--On testing other methods, similar preferential DNA extraction was obtained with Perry and Wolff's FPG method, and with the hot salt pretreatment of Korenberg and Freedlender. However, good Giemsa differentiation could also be obtained using Hoechst 33258 and light pretreatments without any DNA loss. Reverse differentiation patterns (TB pale, BB dark) induced by warm acids resulted in extraction of nearly two thirds of 3H-BrdU label, but relative loss was the same from pale and dark chromatin. Direct reverse staining using alkaline Giemsa did not result in any loss of label.--Thus preferential DNA loss from pale stained chromatin underlies differentiation methods using light plus hot salt pretreatments, but it is not obligatory for good differentiation using other techniques.