A new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture.
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.
Prieto
A
A
Department of Medicine, University of Alcalá, Alcalá de Henares, Madrid, Spain.
Reyes
E
E
Diaz
D
D
Hernandez-Fuentes
M P
MP
Monserrat
J
J
Perucha
E
E
Muñoz
L
L
Vangioni
R
R
de La Hera
A
A
Orfao
A
A
Alvarez-Mon
M
M
D017209:Apoptosis; D002452:Cell Count; D016923:Cell Death; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D066298:In Vitro Techniques; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008853:Microscopy / Q000379:methods; D011786:Quality Control; D012015:Reference Standards