A typical HCI screening project at HiTIF can be subdivided in 5 phases: Pre-development, Subsidy Proposal, Assay Development, Primary Screen and Secondary Screen.

Pre-development: The investigator contacts the Facility Head to inquire about the current availability of instrumentation time and manpower for the project to be run at the HiTIF. At this point the discussion will also involve an initial evaluation of the feasibility of the proposed project taking into account both biological and technological considerations. Typical questions to consider are: can the proposed imaging assay be carried out on tissue culture surfaces? Which is the most appropriate combination of fluorophores for the assay? Can the phenotype of interest be detected by confocal microscopy, and is it suitable for High-Throughput Imaging (HTI)? In case of screening projects, can the chosen cell line be efficiently transfected with oligo RNA? Are there appropriate biological and technical positive and negative controls for the assay? Is the assay window between positive and negative controls known? The availability of positive and negative controls and preliminary results in a low-throughput format, preferably including fluorescence microscopy data, will be essential to assess the feasibility of the project in a miniaturized, high-throughput format. During these initial discussions the HiTIF will provide guidance on how to design the HCI assay in exam.

OSTR Subsidy proposal: HiTIF CCR Collaborators can apply to the CCR Office of Science and Technology Resources Supplemental Technology Award Review System (STARS) for an up to 50% subsidy to the project, for a maximum projected total cost of $20,000 ($10,000 subsidy). STARS submissions instructions can be found here. The proposal is evaluated by an external panel of scientific experts in CCR. Upon subsidy approval, the Assay Development phase of the project can commence. The investigator will be responsible to cover directly for imaging, reagents and consumables charges incurred during the project. Upon completion of the project, the OSTR will refund the HiTIF charges to the investigator up to the total amount originally approved. For projects whose projected budget is above $20,000 the investigator is encouraged to request funds through a regular Resources Request System (RRS) with CCR.

Assay Development: The HiTIF will train, advise and help in the use and troubleshooting of liquid handling and HTI instrumentation. Optimal cell growth conditions and confluence for the experimental conditions to be used during the screening phase will be optimized first in 384- well format, and if needed in 96-well format, in case the former format is not compatible with the assay. Image acquisition,  siRNA or sgRNA transfection conditions will then be optimized. Based on the biological question addressed by the assay, HiTIF personnel will design custom image analysis pipelines to extract relevant quantitative phenotypic cellular features. Quality control descriptors (Z-score, S/N ratio, assay window) based on relevant positive and negative controls will be used to guide the optimization process. Multiple test plates with a sufficient number of positive and negative controls will be run on independent days to determine the inter-plate and inter-day assay variability. Finally, a pilot screen with a library of ~ 300 reagents will be run to test, and possibly troubleshot, the cell seeding, cell culture reagent dispensing, automated image analysis and secondary data analysis workflow in a realistic screening scenario.

Primary Screen: Depending on the size of the libraries of reagents to be screened, primary screens will be run in multiple replicates on independent days. QC Filtering and hit-selection criteria based on biological potency and specificity will be determined by HiTIF personnel in concert with the investigators.

Secondary Screen: Based on the hit-list from the primary screen and on logistical  considerations, a number of target genes or compounds will be selected for secondary screen to rule out off-target effects. 

Last updated by Pegoraro, Gianluca (NIH/NCI) [E] on Aug 30, 2022