Cell-based assays, particularly phenotypic and high-content assays, are gaining importance for chemical biology and drug discovery. Good practice in assay development not only for screening but also for bench scale experiments in any research lab should aim for robust and reproducible assays to address biological questions and receive accurate and trustable data. For cell-based assays many variables accumulate, and components should be carefully chosen. Material selection and its quality are critical. An appropriate cell model has to be chosen (Fig. 1) and cell identity confirmed. Most labs are equipped to allow immediate down-scaling of experiments to 96-well microtiter plates. Multiple wells for one experiment allow replicates and do not limit the number of controls. Excel may be used to elaborate the protocol, list all pipetting steps and prepare pipetting schemes in table format (Fig. 2).
Fig. 1. The advantages of cell lines and questions to consider to identify the right one for the desired biological question and assay type. (This slide is part of the 1-day Short Course 'High-Content Screening', Module 'Assay Development', held at various SBS and SLAS conferences, e.g. ref. 1)
Commercial versus own assays
If own assays are developed, they can be designed specifically to the needs and might be much cheaper. On the other hands, significant time and resources are needed to design, develop and validate an assay, and eventually it might be difficult to scale it up for screening. For many applications, therefore, growing numbers of commercial assays are available from various vendors. Those kits are typically straight-forward and there is a good chance to reproduce the assay in one’s lab. Further, validated cell lines such as reporter cell lines or such with target proteins of interest fused to a fluorescent protein might serve the need already, or serve as starting points for own further assay development. Also validated antibodies are available that have been tested already to work for special applications such as immunostaining in fixed cells or for FACS assays.
During assay design, the question of appropriate positive and negative controls occurs. In most cases ‘untreated’ is seen as appropriate, however, ‘mock’ controls that contains the carrier or solvent, such as DMSO, may be considered as more appropriate. A non-target or inactive control (e.g. an inactive variant of the positive control compound) might be an option as well. The best positive controls are functional, target-directed and assay-specific controls. However, such might be not available.
Fig. 2. Working in 96-well microtiter plates offers numerous advantages. (ref. 1, adapted from ref. 2)
During assay development numerous steps, conditions and parameters are systematically optimised: from cell density and plating conditions, compound addition, incubation time, washing and staining conditions, till optimising the reading conditions. For cell-based assays never forget that the principle tool of a cell biologist is the microscope! All steps and processes can be best observed by eye, performance assessed and improvements considered. An important improvement might be to simplify the protocol and try to combine steps, particularly in lengthy protocols that have been elaborated over generations such as immunostaining.
Finally, the assay should be well characterised with the desired set of controls in repeated experiments (Fig. 3). What is ‘normal’ or ‘background’?, and how variable is that? What is the variability of the desired phenotype? How reproducible is that and what is its variance? What is the signal window, the difference between desired phenotype over normal or background? What is the variance with-in a plate, between plates of the same experiment, and between independent experiments. A helpful way to analyse and assess the quality of the assay might be the Z’-factor determined by Zhang and colleagues (ref. 4; Fig. 4)
Fig. 3. Assessment of an assay: Describe variability and signal window. (ref. 1, adapted from ref. 3)
Fig. 4. Assessment of an assay: Determination of variation, assay window and Z’-factor (ref. 1, adapted from ref. 3; 4, 5)
(1) Krausz, E., Evans, J.G., Lemmon, V., Schuerer, S.: SLAS 1-day Short Course ‘High-Content Screening: Instrumentation, Assay Development, Screening, Image and Data Analysis’. SLAS 1 st Annual Conference and Exhibition, Society for Laboratory Automation and Screening, February 4-8, 2012, San Diego, California, USA.
(2) Krausz, E.: 96-well Electroporation for Plasmids and Oligonucleotides. amaxa's Workshop on RNAi and Nucleofection, September 11, 2007, Dresden, Germany.
(3) Krausz, E.: Assay Design, Development & Validation. RNA Interference & High-Content Screening Applied to Target Discovery, RIGHT Symposium, September 11-12, 2006, Dresden, Germany.
(4) Zhang, J.H., Chung, T.D., Oldenburg, K.R.: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999, 4:67-73.
(5) Krausz, E., Grabner, A., Kroenke, A., Sachse, C., Echeverri, C.J.: Optimising high throughput RNAi-based assays using transient transfection of synthetic siRNAs in cultured mammalian cells. In Engelke D.R. (ed): RNA Interference (RNAi)- Nuts & Bolts of RNAi Technology, p. 131-168, DNA Press LLC, (2003).
Recommended further reading:
Fav a, E., Krausz, E., Barsacchi, R., Baines, I., Zerial, M.: High-Content Phenotypic Cell-Based Assays. In Shorte, S.L., Frischknecht, F. (eds.): Principles and Practice: Imaging Cellular and Molecular Biological Functions, p. 423-442, Springer-Verlag ( Berlin Heidelberg, Germany), (2007). http://books.google.de/books?id=p-HyDWUhDS0C&pg=PA423&lpg=PA423&dq=eberhard+krausz&source=bl&ots=45YT2k5ZhS&sig=n4e4VOEzRYSRrekpJyi0StyNxXM&hl=de&sa=X&ei=7CUPUt3GFOev7Qa834D4CA&ved=0CFoQ6AEwBzge#v=onepage&q=eberhard%20krausz&f=false
Chakravarti, A., Bowman, D., Ecsedy, J.A., Rabino, C., Donovan, J., D’Amore, N., Veiby, O.P., Rolfe, M., Das, S.: Developing Robust High Content Assays. In Haney, S.A. (ed.): High Content Screening: Science, Techniques and Applications, p. 85-110, Wiley ( Hoboken, New Jersey), (2008).
Assay Guidance Manual, Edited by G. Sitta Sittampalam, http://www.ncbi.nlm.nih.gov/books/NBK53196/
Dr. Eberhard Krauß
www.chembiocon.de, Wiesbaden, Germany
www.chembiocon.com, Brussels area, Belgium