Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures.
BACKGROUND
Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI).


METHODS
This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture.


RESULTS
In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells.


CONCLUSIONS
This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.
Prieto
Alfredo
A
Department of Medicine, CSIC R&D Associated Unit University of Alcalá, Alcalá de Henares, Madrid, Spain.
Díaz
David
D
Barcenilla
Hugo
H
García-Suárez
Julio
J
Reyes
Eduardo
E
Monserrat
Jorge
J
San Antonio
Esther
E
Melero
David
D
de la Hera
Antonio
A
Orfao
Alberto
A
Alvarez-Mon
Melchor
M
D017209:Apoptosis / Q000502:physiology*; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology; D002452:Cell Count / Q000379:methods*; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000166:cytology / Q000502:physiology; D008214:Lymphocytes / Q000166:cytology*; D013601:T-Lymphocytes / Q000166:cytology / Q000502:physiology
D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D004247:DNA