A new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture

Background: Interval standards hare been used in flow cytometry methods to enumerate lymphoid subsets and hemopoietic progenitor cells ex vivo. Hoc-ev er, the currently available methods cannot be readily applied to the analys is of cultured cells because of the frequent occurrence of cell death durin g in vitro assays. Methods: This paper reports a new method for the enumeration of both viable and nonviable cells in culture. Cells were counted with the aid of an inte rnal reference standard of microbeads, and live versus dead cell discrimina tion was performed using 7-amino-actinomycin D which allows the double stai ning of surface antigens. Results: The method is more precise, accurate and sensitive than either con ventional Light microscopy-based or automated cell counting. Additionally, it may be used to accurately measure the number of apoptotic cells in a cul ture. Results: Through the enumeration of surviving cells it is demonstrate d that, when applied to the study of mitogen-activated T lymphocytes, curre nt flow cytometry techniques (which do not use internal standards) for the study of the viability and apoptosis overestimate the fraction of viable ce lls and underestimate both the fraction of dead and apoptotic cells. Conclusions: The new method overcomes these limitations and is of use in th e in vitro study of cell growth and apoptosis. (C) 2000 Wiley-Liss, Inc.