Table 1.

Biochemical and molecular biology tools for HCS: summary of their application, advantages, and potential pitfalls.

Assay tools	Applications	Advantages	Potential pitfalls	Selected References
IHC	Quantification of drug target proteins Identification of subcellular localizations Phenotypic characterization Cell-ECM interaction studies	Detection of endogenous protein levels Detection of phosphorylated proteins Antibodies readily available for a multitude of antigens Multiplex protein detection No requirement of gene delivery	Laborious procedures requiring optimization False positives based on antibody quality Background signals from non-specific antibody binding Terminal assay, not real-time	[31] [118] [50] [95]
RNAi screen	Functional gemomics through genome-scale loss-of-function screens Validation of new drug targets Therapeutic drug repositioning	Non-terminal/invasive assay RNAi libraries readily available No requirement of generation of stable cell lines Large-scale, phenotype-based screens	Variability and degree of knockdown Only temporary inhibition of gene function Potential non-specificity Not every gene is susceptible to RNAi	[11] [24] [49] [61, 62] [9] [78]
RGA	Studies for gene expression and regulation Phenotypic characterization Mechanism-based toxicity assays Studies relating to cell-cell and cell-ECM interactions	Non-terminal assay Compatible with time-lapse studies Amenable to modifications such as signal amplification and gene delivery method Sensitive, rapid, and reproducible	Limitation of multiplex protein detection Cell-to-cell variability in expression can confound results Indirect measurement of expression Poor transfection efficiencies Laborious procedure to generate stable cell lines Reporter systems cannot accurately capture complexity of gene regulation	[39] [6] [23] [27] [68] [46] [47]
CRISPR-Cas9	Functional genomics Phenotypic characterization Quantification of target proteins	Non-terminal assay Detection of endogenous protein levels Fewer false signals Compatible with time-lapse studies Large-scale, phenotype-based screens	Laborious protocols for isolation of successfully targeted cells Current lack of arrayed screening resources (e.g., genome-scale collections of sgRNAs)	[99] [109] [7] [93] [119]
PPI	Identification of protein-protein interactions Identification of small-molecule-targeted pathways Identification of optimal targets for a network of interest	Non-terminal assay Compatible with time-lapse imaging Detection of phosphorylated proteins Robust and high specificity Direct detection of molecular interactions Detection of subcellular localizations and translocations of protein complexes	Indirect measurement Requirement of stable cell lines or high transfection efficiency Non-specific protein binding leading to false signals	[96] [102] [107] [75] [48] [1] [90]