A multiplexed assay describes an experiment that measures many parameters from a single study, which has been set up within a well. These assays are commonly employed to obtain mechanistic insights into biological and toxicological processes.
The ability to study two or more parameters at one time point on the same cells increases throughput and reduces the costs by minimizing cell culture and consumable requirements, as well as reducing the time required for set-up.
Although highly beneficial, the development of robust multiplexed assays for compound screening can prove technically demanding and time-consuming. Important technical considerations such as choice and concentration of labelling dyes (to ensure they are spectrally distinct), compatibility of assay chemistries and buffers, choice of instrumentation for image analysis, and scanning and software analysis need to be addressed.
With a wide range of fluorescent dyes available today and instruments that can obtain multiple data rapidly from a single scan, multiplexed assays can be a worthwhile investment in a high-throughput screening environment. However the need to adequately and carefully plan and set up preliminary assays is vital to ensure robust and valuable results. The tips below aim to help initial set-up of multiplexing assays for high-throughput imaging.
- Choose a combination of fluorophores with minimal spectral overlap.
- Using known positive and negative control compounds separately, optimize the assay conditions to ensure specific detection responses for each experimental parameter (i.e., the optimal inhibitory concentration of both the compound and fluorophores required for the assay).
- Perform titrations in separate wells with separate fluorophores for the separate parameters of interest, and overlay or combine scan results. Identify fluorophore/reagent concentrations that produce minimal spectral overspill while maintaining good assay results.
- Assess the effect of combining two or more fluorophores per well to allow a two parameter (duplex) measurement over a concentration range of the control compound. This may require additional reagent concentration optimization, and the assay performance for the individual parameters may have to be compromised to allow compatibility with multiplex format. (If this is not easily achievable it may be worth considering alternative fluorophores hence returning to stage 2.)
- Once satisfied with the multiplexing capabilities of these fluorophores for the control compounds, it is wise to validate this assay on a range of other known inhibitory compounds to ensure correct responses.
In summary, multiplexing can be a useful tool for the study of the effect of compounds on multiple cellular parameters in high-throughput imaging. With careful planning, experimental development, and effort, this style of assay coupled with fast single-step scanning instruments can provide robust, data-rich results in a time- and cost-efficient manner.