DOUBLE LABELING OF S-PHASE MURINE CELLS WITH BROMODEOXYURIDINE AND A SECOND DNA-SPECIFIC PROBE

Abstract

A rapid method was developed which combines immunofluorescence and autoradiography and permits the double labeling of DNA. P388 murine leukemic cells were incubated with bromodeoxyuridine and 3H-thymidine simultaneously. After fixation, the sample was 1st processed with a monoclonal antibody to bromodeoxyurine (RPMB I) so that any cell in S-phase was brightly fluorescent (RPMB technique). Next 3H-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 fluoresence and grains per cell and the data demonstrated that the number of [3H]ara-C grains in each P388S cells 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 was introduced which will be useful to a wide variety of researchers because this technique together with the digital analysis system offers the possiblity of measuring drug sensititivites in individual cells.