A. Prieto et al., A new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture, CYTOMETRY, 39(1), 2000, pp. 56-66
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.