The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells.
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.
Al-Nasiry
S
S
Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium. salwan.alnasiry@gmail.com
Geusens
N
N
Hanssens
M
M
Luyten
C
C
Pijnenborg
R
R
D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival; D002822:Choriocarcinoma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D009361:Neoplasm Invasiveness; D010078:Oxazines / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
D010078:Oxazines; D016212:Transforming Growth Factor beta; D014966:Xanthenes; C005843:resazurin