Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout.
We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.
Schreer
Anja
A
Department of Cell Toxicology, UFZ Centre for Environmental Research, Leipzig-Halle in the Helmholtz Association, 04318 Leipzig, Germany.
Tinson
Cheryl
C
Sherry
James P
JP
Schirmer
Kristin
K
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005452:Fluoresceins / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology*; D017686:Oncorhynchus mykiss; D010078:Oxazines; D010416:Pentachlorophenol / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D014819:Vitellogenins / Q000096:biosynthesis; D014966:Xanthenes
C505935:5-carboxyfluorescein diacetate acetoxymethyl ester; D000199:Actins; D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010078:Oxazines; D012333:RNA, Messenger; D014819:Vitellogenins; D014966:Xanthenes; C005843:resazurin; D004958:Estradiol; D010416:Pentachlorophenol