Assay plates:
Black-walled, clear-bottom (plastic or glass) microplates are used for image-based screens. A common 384-well plate type is Corning #3764 (previously #3712).
When imaging at higher magnifications, we recommend Revvity PhenoPlates (eg #6057302) or equivalent.
Seeding density:
Test a variety of cell densities under the conditions you plan to use for the screen. Image analysis is often improved when the cells are sub-confluents. Depending upon cell size and length of assay, a starting range may be 500 to 2,000 cells per well.
Imaging live cells:
This is possible when using the Acumen, but the the stage is not heated and your cells will be at room temperature during imaging.
Treating plates after imaging:
After processing cells for imaging, fill your wells with PBS to prevent the cells from drying out and to reduce background noise. Some investigators include an anti-fungal agent (eg sodium azide) for long term storage at 4oC.
Selecting fluorophores:
Please make sure the fluorophores you select do not conflict with the filter sets on the screening scope. See details for each microscope. For high throughput screens, antibodies are frequently used at higher concentrations to decrease acquisition time.
Plate Seals:
Clear and foil microplate seals can significantly increase background fluorescence to the point where images may be unusable. Black opaque plate seals are ideal for fluorescence imaging. Microplates must be sealed prior to imaging in the microscope to protect the equipment.
