Although a successful cell-based assay depends on the integration of several essential elements, it can be argued that the cell culture model system is the most important component. If the model system does not reflect the relevant biological event, the assay chemistry and instrumentation cannot compensate. If the culture model is a good representation of events to be measured, it will provide more flexibility in the choices of assay chemistry and instrumentation used. A number of questions should be addressed during development of cell-based assays to measure viability or apoptosis to ensure the model system represents a physiologically relevant event and will provide meaningful results. The relevant questions include:
• What type of cells are the best predictors of human toxicity?
• What culture conditions can produce enough cells for screening?
• Should antibiotics be used for stock culture of cells or during assay?
• What sample density (96-, 384-, or 1536-well) should be used?
• How many cells per well are used for different plates?
• Which robotic liquid handlers can dispense live cells?
• When should the test compound be added relative to plating cells?
• What concentration of compound should be tested?
• Should more than one concentration of test compound be assayed?
• How much DMSO can be tolerated by cells in the assay?
• Does each assay plate need positive and negative controls and is placement on the plate critical?
• How long should cells be exposed to the test compound before recording viability?
• Will the assay chemistry affect the results?
• Should the same assay be repeated to confirm "hits" or should a different method be used?
• Can multiplexing more than one assay be done simultaneously to increase efficiency?
Initially, the list may seem overwhelming; but investment of time up front to consider these issues during assay development will decrease the occurrence of artifacts and false hits. The answers to all of these questions will depend on the in vitro model system chosen.
Was this article helpful?