Use of a double-label method to detect rapid changes in the rate of cell proliferation.

We developed a double-label method to directly measure the rate at which cells enter S-phase of the cell cycle. All cells in S-phase were first labeled with a short pulse of 3H-thymidine. This was followed by a longer incubation in bromodeoxyuridine (BrdU), a thymidine analogue. Nuclei labeled with 3H-thymidine were detected by autoradiography and those labeled with BrdU by immunocytochemistry. Cells labeled only with BrdU must have entered S-phase at some time after the end of the 3H-thymidine pulse. Thus, the rate of entry of cells into S-phase could be determined. This method was shown to be more accurate and more sensitive than determining changes in the rate at which cells entered S-phase with a continuous labeling protocol. It was possible to detect changes in proliferative activity that occurred in less than 1 hr. We used this double-label technique to study changes in the cell cycle during the terminal differentiation of chicken embryo lens fiber cells. These studies revealed differences in the effects of several treatments known to stimulate fiber cell differentiation. They also demonstrated the presence in the embryonic eye of factors that stimulate and prevent lens cell proliferation and differentiation.