title: High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.
abstract: GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid β-galactosidase (β-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1(-/-) cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes.
authors.affiliation: BioStrategies LC, State University, P.O. Box 2428, AR 72467, USA.
authors.forename: Walter
authors.initials: W
authors.lastname: Acosta
authors.affiliation: BioStrategies LC, State University, P.O. Box 2428, AR 72467, USA; Arkansas Biosciences Institute & Department Biological Sciences, Arkansas State University, P.O. Box 639, Jonesboro, AR 72467, USA.
authors.forename: Reid
authors.initials: R
authors.lastname: Martin
authors.affiliation: BioStrategies LC, State University, P.O. Box 2428, AR 72467, USA.
authors.forename: David N
authors.initials: DN
authors.lastname: Radin
authors.affiliation: BioStrategies LC, State University, P.O. Box 2428, AR 72467, USA; Arkansas Biosciences Institute & Department Biological Sciences, Arkansas State University, P.O. Box 639, Jonesboro, AR 72467, USA.
authors.forename: Carole L
authors.initials: CL
authors.lastname: Cramer
keywords: Acid β-galactosidase; Cholera toxin B subunit; GM1-gangliosidosis; High-throughput imaging