Document Detail


Mathematical analysis of mis-estimation of cell subsets in flow cytometry: Viability staining revisited.
MedLine Citation:
PMID:  21362427     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labelled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behaviour. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis.
Authors:
A M Petrunkina; R A P Harrison
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-2-26
Journal Detail:
Title:  Journal of immunological methods     Volume:  -     ISSN:  1872-7905     ISO Abbreviation:  -     Publication Date:  2011 Feb 
Date Detail:
Created Date:  2011-3-2     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2010. Published by Elsevier B.V.
Affiliation:
Unit of Reproductive Medicine of Clinics, University of Veterinary Medicine Hannover Foundation, Clinic for Horses; Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
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