Application of a DNA double labelling method for the flow cytometric analysis of recruitment of non-cycling cells in a mixed population of P and Q cells

Abstract

In this paper we describe the application of a non-radioactive DNA double labelling and staining method to an analysis of cell proliferation kinetics by flow cytometry, aimed at the direct measurement of recruitment rates in cell cultures. The method is based on the application of two halogenated deoxyuridines: iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) which are incorporated into DNA synthesizing cells. By applying two commercially available monoclonal antibodies both deoxyuridines can be detected separately. To measure recruitment all proliferating cells in a plateau phase culture were labelled first with IdUrd applied during a time interval approximately equal to the cell cycle time. Subsequently, recruitment induced by a medium change was analysed by flow cytometric assessment of incorporation of CldUrd in cells which had not taken up IdUrd. Experiments designed to determine the toxicity of continuous labelling with IdUrd in different concentrations and of pulse labelling with CldUrd showed that there was no effect on the progression of cells through the cell cycle. The aim of this study is to test the sensitivity of the procedure to detect changes in proliferation kinetics, in particular the entrance of resting cells into the S phase. Although the cell culture model used is very simple, the results demonstrate clearly that a low rate of recruitment can be detected. It is suggested that the procedure described here is specific and sensitive enough to quantify changes in cell proliferation in tumours induced by various treatments and has advantages over other methods, which measure recruitment indirectly, or directly by using two radioactive thymidines.