Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement.
Current in vitro assays used in assessing tumor motility could be improved by the development of a simple technique that would facilitate studies of the impact of specific genes on pharmacologically altered chemotaxis. We developed a technique that improves on the classic transwell assay by using fluorescence and luminescence to assess chemotaxis. In this transient transfection system, co-transfection of a reporter construct and a gene with an unknown impact on motility are coupled with biochemical assays to quantitate the number of cells that have received a transferred gene, which subsequently crosses the membrane. This assay was found to be less variable than the conventional transwell chamber and is easily adaptable to studies of cell motility or cell invasion. We also demonstrate that this assay can detect the effect of both genetic and pharmacological inhibition of motility alone and in combination. It therefore has the potential to reveal additive or synergistic effects.
Gildea
J J
JJ
University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Harding
M A
MA
Gulding
K M
KM
Theodorescu
D
D
D002452:Cell Count; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement; D002633:Chemotaxis / Q000235:genetics; D005453:Fluorescence; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D008156:Luciferases / Q000032:analysis*; D009361:Neoplasm Invasiveness; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000473:pathology*
D005456:Fluorescent Dyes; D008156:Luciferases