Methods to increase the capacity of high content cell-based screening assays

A method for increasing the throughput of high content cell based screening assays, comprising:

• a) providing at least a first array of locations that contain multiple cells, wherein the cells comprise a first reporter set, wherein the first reporter set comprises at least a first fluorescent reporter molecule and a second fluorescent reporter molecule, wherein the fluorescent reporter molecules in the first reporter set
• i) report on different cellular events;
• ii) exhibit phenotypically similar behavior;
• and
• iii) emit fluorescence at wavelengths detectable in at least a first channel of a fluorescence detection device;
• b) conducting a primary screen that comprises imaging the at least first array of locations in high content mode to obtain fluorescent signals in the first channel from the fluorescent reporter molecules in the first reporter set, wherein the imaging occurs either before, after, and/or simultaneously with contacting of the at least first array of locations with one or more test compounds;
• c) detecting test compound induced changes in the fluorescent signals from the fluorescent reporter molecules in the first reporter set, wherein a test compound induced change in the fluorescent signals indicates an effect of the one or more test compounds on one or more cellular events reported on by the fluorescent reporter molecules in the first reporter set;
• and
• d) deconvolving the test compound induced changes, wherein the deconvolving comprises conducting one or more secondary screens, wherein at least one of the secondary screens comprises a method selected from the group consisting of:
• i) screening positive locations on the first array of locations in which test compound induced changes were detected in the primary screen, and optionally screening one or more control locations on the first array of locations, wherein the cells in the positive locations and any control locations to be screened in the secondary screen are further contacted with two or more further fluorescent reporter molecules that are optically distinguishable from each other and from the fluorescent reporter molecules in the first reporter set, wherein at least one of the further fluorescent reporter molecules reports on the same cellular event as the first fluorescent reporter molecule, and wherein at least one of the further fluorescent reporter molecules reports on the same cellular event as the second fluorescent reporter molecule;
• and
• ii) screening an at least second array of locations and an at least third array of locations that contain multiple cells, wherein the cells on the at least second array of locations comprise the first fluorescent reporter molecule and not the second fluorescent reporter molecule;
• and wherein the cells on the at least third array of locations comprise the second fluorescent reporter molecule and not the first fluorescent reporter molecule;
• wherein the deconvolving comprises contacting the at least second array of locations and the at least third to array of locations with only those test compounds that produced test compound induced changes during the primary screen, and optionally with one or more control compounds.