Multiplexing high-throughput imaging assays offer researchers a powerful means for running cell-based screens, measuring the levels of multiple targets/readouts at the same time and on the same sample of cells.
The results of these assays provide valuable mechanistic and quantitative insights into biological and toxicological processes, paving the way to important discoveries. But this isn’t the only reason to multiplex; this tool can also reduce costs by maximizing the research scientist’s time (decreasing the amount of time required for setup) and minimizing consumable requirements.
Today, there is a wide range of fluorescent dyes available and advanced high-content imaging instruments that can rapidly obtain multiple data sets from a single scan. Consequently, multiplexed assays offer a valuable, cost-effective approach for the high-throughput screening laboratory.
Key considerations
Before embarking on a new screen, take some time to consider the following. As with so many research endeavors, thorough preparation is the key, and you must first have a proficient knowledge of the technical elements that go into developing robust multiplexed assays for high-content screening.
For example, to achieve the highest-quality results, you must select the right instrument for image capture and analysis and know how to choose the correct types and concentrations of labeling dyes for the imaging instrument you are using.
Given these considerations and the extra time required to develop the assays, is multiplexing worth the effort? It certainly is. Just be sure to carefully plan and set up your preliminary assays, taking into account the following guidelines:
1. Choose a combination of fluorophores with minimal spectral overlap. This ensures that the images you capture for each fluorophore have no spectral spill, making image analysis simpler and more reliable.
2. Optimize your assay across all variables. Start by using only known positive and negative control compounds separately, and test your assay conditions - cell type, seeding density and compound incubation time - to explore the range of detection responses for each experimental parameter. This will vary from assay to assay, depending on the nature of the compound being screened, the type of cell in use, the concentrations involved and many other variables. Therefore, it is essential to identify the ideal parameters for your specific screen, via pilot studies, before starting the full experiment.
3. Test your fluorophores to make sure they work well together. Multiplexing requires that two (or more) assays work under similar experimental conditions (i.e., in the same well). Therefore, multiplexed-assay optimization may require additional reagent-concentration optimization, and the assay performance for the individual parameters may need to be somewhat compromised to enable compatibility with multiplexing. If this is not easily achievable, it may be worth considering alternative fluorophores.
4. Carry out fluorophore titrations. The next step is to titrate your different fluorophores, selected for the parameters of interest, in separate wells. You can then overlay or combine your scan results. The resultant data allow you to identify the fluorophore/reagent concentrations that will produce minimal spectral spill while still providing insightful assay results.
5. Validate your assay for broader application. When you’re satisfied with the multiplexing capabilities of these fluorophores for the control compounds, it is advisable to test your new experimental setup on several target compounds to validate its wider-scale accuracy and assay stability.
6. Progress to final screening. You can now carry out your full screening study, safe in the knowledge that your pilot assays have given you the best possible chance of success.
With careful planning and experimental development, multiplexed assays can provide robust, data-rich results in a time- and cost-efficient manner. This is especially true when coupled with fast, single-step, high-content imaging instruments that can image multiple fluorophores simultaneously and are therefore optimized for rapid, high-throughput analysis.
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