Open AccessA new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture
Author(s) -
Prieto Alfredo,
Reyes Eduardo,
Diaz David,
HernandezFuentes María P.,
Monserrat Jorge,
Perucha Esperanza,
Muñoz Leticia,
Vangioni Ricardo,
de la Hera Antonio,
Orfao Alberto,
AlvarezMon Melchor
Publication year - 2000
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(20000101)39:1<56::aid-cyto8>3.0.co;2-v
Subject(s) - flow cytometry , apoptosis , biology , cell counting , programmed cell death , cytometry , haematopoiesis , microbiology and biotechnology , enumeration , progenitor cell , in vitro , cell culture , stem cell , cell cycle , biochemistry , mathematics , genetics , combinatorics
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. RESULTS 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 mitogen‐activated 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. CONCLUSIONS 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 © 2000 Wiley‐Liss, Inc.