Double labeling of S-phase murine cells with bromodeoxyuridine and a second DNA-specific probe

A rapid method has been developed which combines immunofluorescence and autoradiography and permits the double labeling of DNA. P388 murine leukemic cells were incubated with bromodeoxyuridine and tritiated thymidine simultaneously. After fixation, the sample was first processed with a monoclonal antibody to bromodeoxyuridine (RPMB I) so that any cell in S-phase was brightly fluorescent (RPMB technique). Next, tritiated thymidine grains were developed by autoradiography, and the result demonstrated fluorescence as well as black grains in each S-phase cell. P388 cells sensitive (P388S) and resistant (P388R) to 1-beta-D-arabinofuranosylcytosine (ara-C) were incubated with bromodeoxyuridine and [3H]ara-C simultaneously. Processing by autoradiography and RPMB techniques revealed that all S-phase cells in the P388S sample demonstrated vivid "double labeling," whereas P388R cells only revealed bright green fluorescence in S-phase cells, but no grains, confirming a lack of ara-C incorporation into the DNA by this line. Finally, a computerized digital analysis system attached to a microphotometer was used to quantitate fluorescence and grains per cell, and the data demonstrated that the number of [3H]ara-C grains in each P388S cell was inversely proportional to the degree of fluorescence in that cell, indicating that DNA synthesis was inhibited by ara-C. In conclusion, a simple, easy-to-use double-labeling method has been introduced which will be useful to a wide variety of researchers, because this technique together with the digital analysis system offers the possibility of measuring drug sensitivities in individual cells.