Target identification and validation
Identification of therapeutic targets in basic research is often done using CRISPR or RNAi-based screens. Once the possible hits have been identified, flow cytometry is useful to validate the phenotype of each hit after a knockout, knockout, or down. Flow cytometry is particularly useful for testing the phenotype of suspended cells, such as immune system cells, although yields have been limited in the past, making large-scale studies using this method impossible.
High-throughput flow cytometry systems are becoming increasingly available, including instruments with automatic sample plate loading, significantly reducing run time (and overall time). Additionally, advances in automated sampling and reduced sampling volume have resulted in larger screens. Smaller sample sizes reduce the number of reagents needed for testing and cost and allow the use of more easily translatable cell-based models. Current cytometry can measure several parameters simultaneously so that complex phenotypes can be detected, facilitating the identification of new mechanisms. In addition to the physical aspect of loading and obtaining samples, data collection and processing can be limiting as most systems are designed for small-scale laboratory use rather than conducting large-scale testing. Researchers should look for advanced flow cytometry platforms with built-in data analysis that meet the needs of their specific test.
Antibody Library Screening
Once potential targets have been identified as described above, therapeutic agents should be developed if there are no targets. If the therapy is antibody-based, the appropriate antibodies must be identified to bind to the target. This can be achieved using animal imaging or immunization technologies.
Technology-based screen capture
Flow cytometry can help identify which of an existing library of antibody or antibody-like molecules has sufficient specificity, selectivity, and affinity for the antigen. With the ability to measure multiple fluorophores simultaneously, flow cytometry allows for multiplexing by combining positive and negative cell lines and adding “off-target” cell lines to a single experimental well. Collecting data on specificity and selectivity is therefore achieved with minimal sample input. Each cell line is identified by a specific fluorescent marker so that the antibody that binds to each cell type can be distinguished and quantified.