✍ Scribed by Alfredo Prieto; Eduardo Reyes; David Diaz; María P. Hernandez-Fuentes; Jorge Monserrat; Esperanza Perucha; Leticia Muñoz; Ricardo Vangioni; Antonio de la Hera; Alberto Orfao; Melchor Alvarez-Mon
No coin nor oath required. For personal study only.
Background: Internal standards have been used in flow cytometry methods to enumerate lymphoid subsets and hemopoietic progenitor cells ex vivo. However, the currently available methods cannot be readily applied to the analysis of cultured cells because of the frequent occurrence of cell death during 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 internal reference standard of microbeads, and live versus dead cell discrimination was performed using 7-amino-actinomycin D which allows the double staining of surface antigens.
The method is more precise, accurate and sensitive than either conventional light microscopy-based or automated cell counting. Additionally, it may be used to accurately measure the number of apoptotic cells in a culture.
Results: Through the enumeration of surviving cells it is demonstrated that, when applied to the study of mitogenactivated T lymphocytes, current flow cytometry techniques (which do not use internal standards) for the study of the viability and apoptosis overestimate the fraction of viable cells and underestimate both the fraction of dead and apoptotic cells.
The new method overcomes these limitations and is of use in the in vitro study of cell growth and apoptosis. Cytometry 39:56 -66, 2000.
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