HTS - Important Considerations

Assay type

• Biochemical assays
• Cell-based assays (2D, 3D spheroids, organoids)

Readout/measurement type

• High-content (microscopy)
• Spectroscopic (fluorescence, luminescence, absorbance)

Plate format

• Plate type 96- or 384-well format
• Plate color: transparent, white, black
• Well shape: flat, round, V-shaped
• Plate material: polystyrene, polypropylene, glass, etc.
• Coating: none, TC, ultra-low attachment, etc.

Controls

• Selection of appropriate negative- and positive controls
• Solvent concentration (e.g. 0.1 % DMSO, max. 0.3 % for cell-based assays)
• Number of controls and replicates

Assay Quality

• Z’-factor: measure of assay quality based on the lower and upper limit of the assay and the standard deviations of the positive and negative controls.
• Z’ value 0.5-1.0: excellent assay
• Z’ value 0.0 - 0.5: poor to acceptable assay
• Z’ value < 0.0: assay not working since positive and negative controls overlap

Handling capabilities of the equipment

• Minimum volumes
• Dead volumes
• Cooling/heating of media or reagents
• Edge effects

Assay Data Analysis

• Correction of edge effects and cell seeding patterns
• Normalization
• Hit selection

Data storage

• Imaging-based assays require large storage capacity compared to spreadsheet-based data