Table 2.

Cell-Based Assays

Cell-Based Assays	Requirements	Pros/Cons
Fitness/viability (growth rate, competitive fitness, drug suppression, cell titer-glo)	Protein of interest must be involved in a process with clear functional impact Wild type and sector mutant cells (can be arrayed or pooled and barcoded) Growth/viability monitoring	Quantitatively determines phenotypic consequence of mutations Can be performed in medium- and high-throughput Allows detection of small (or at least physiologically relevant) effects Requires obvious functional readout for protein
Morphology and subcellular localization (differentiation, yeast mating projection, live-cell or immuno-fluorescence)	Wild type and sector mutant cells (separated) Imaging by microscopy	Visually-convincing phenotype Only applicable to certain proteins Low throughput – each mutant must be independently assayed
Transcriptional reporter (fluorescent reporter of a kinase pathway or stress response)	Wild type and sector mutant cells expressing the reporter (can be arrayed or pooled and barcoded) Flow cytometry or other readout for reporter activity	Quantitatively determines consequence of mutations Can be performed in medium- and high-throughput Requires reporters for specific signaling pathways
Phosphorylation (western blot, IF, flow cytometry)	Wild type and sector mutant cells (separated) Antibodies to monitor phosphorylated state of protein of interest	Direct readout of signaling pathway activity Only applicable to certain proteins Low throughput – each mutant must be independently assayed Can have low sensitivity/poor signal-to-noise