DiscussionFluorescence remains one of the most popular detection modes used in cell-based assays primarily due to the wide array of fluorophores available. These can be grouped into three main families of fluorophore:
? reactive dyes that can be used to make fluorescent conjugates of biomolecules (i.e. antibodies) that can be used to probe cellular processes
? fluorogenic probes that upon binding undergo significant changes in fluorescence quantum yield
? photoproteins like the family of GFP variants that can be transfected into cells as reporter systems or fusion proteins.
One of the main limitations of fluorescence detection is background fluorescence, either from autofluorescence from endogenous cellular material, impurities from reagents added to the assay, unbound fluorescent conjugates or fluorogenic probes, or untransfected photoprotein. Autofluorescence and background from impurities can be
reduced by the use of red-shifted fluorophores; unbound fluorescent conjugates can be removed through gentle wash cycles that allow cells to remain adhered to plasticware; but for homogeneous assays in microplates that rely on simple mix and read workflows,
background from unbound probe or untransfected photoprotein can be a problem that limits assay performance.
Automated digital fluorescence microscopy with appropriate image analysis software has the ability to ignore much of this background fluorescence and collect only fluorescence signals emanating from the cells and not the bulk solution in the well. In this work will will demonstrate this capability for a GFP reporter system, a phenotypic
assay measuring reactive oxygen species production and a live cell 3D cell culture-based assay using spheroids.
Brad Larson and Peter Banks
BioTek Instruments, Inc., Winooski, Vermont, USA