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Hum. Reprod. Advance Access originally published online on February 16, 2007
Human Reproduction 2007 22(5):1304-1309; doi:10.1093/humrep/dem011
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells

S. Al-Nasiry1,2, N. Geusens2, M. Hanssens, C. Luyten and R. Pijnenborg

Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium

1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. Tel.: +32 486994946; Fax: +32 16344205; E-mail: salwan.alnasiry{at}gmail.com

BACKGROUND: The current techniques for quantifying trophoblast viability, migration and invasion are mainly limited by the need to sacrifice the cells during the test procedure. In this study, the vital dye AB (AB) was used to quantify cell number and viability of BeWo and JEG-3 choriocarcinoma cells, as well as their migration and invasion through fibronectin-coated filters.

METHODS: AB was directly added to culture medium of incubated test and control cells. At various time intervals, the redox reaction, in which AB is reduced by the cells, was measured by absorbance readings at 540 and 630 nm. For cell migration and invasion, cells were cultured onto uncoated or fibronectin-coated inserts, respectively. AB reduction of migrated cells was normalized to that of control cells to calculate percentages of migration. This model was also tested in the presence of a reported inhibitor, transforming growth factor (TGF) beta.

RESULTS: The curve of %AB reduction versus cell number was linear, with intra- and inter-assay Coefficient of Variations of 1.88%and 2.94%, respectively. AB reduction increased with both seeding concentrations and incubation time with AB. TGFbeta treatment caused a modest decrease in AB reduction in both JEG-3 and BeWo cells. TGFbeta treatment also decreased migration in BeWo, but not in JEG-3, cells.

CONCLUSIONS: AB assay is a simple and reliable method for quantifying trophoblast viability, migration and invasion.

Key words: Alamar Blue/choriocarcinoma/invasion/migration/viability

2 Share equal contribution to this article

Submitted on June 24, 2006; resubmitted on December 18, 2006; accepted on December 27, 2006.


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