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University of Colorado Denver


High-Throughput Screening and Chemical Biology Core Facility

HTS and Chemical Biology Information and Resources

A typical HTS campaign encompasses three main steps, outlined below. It is​ assumed that the general concept of the screening approach, as well as the possible assays involved, have been discussed between the PI and the HTS Director. It is also highly preferable, though not absolutely required, to have initial results and, if possible, a working assay in hand prior to initiating the screening effort. This enables the HTS lab to focus immediately on the first phase: Adaptation and statistical validation of the assay or screen. Please click on the steps below for a detailed description.

Step One: Assay Development and Validation

Step Two: Screening

Step Three: Reconfirmation and Follow-Up

Step One: Assay Development and Validation

1. Initial assay questions or parameters that need to be considered and optimized (note that some apply more to biochemical or enzyme assays, while others are more important for cell-based or high-content screens):

  1. Can the assay (plates) be read using standard HTS lab equipment?
  2. Assay Parameters:
    1. DMSO tolerability: Critical parameter. All test compounds are stored in DMSO. Standard compound screening concentration is 10uM, and the compound libraries are stored at 2mM in DMSO, which leaves a residual DMSO concentration of 0.5% in assays. A DMSO tolerability curve between 0% and at least 1% DMSO is a critical step.
    2. Linear range of the reaction/response
    3. Linear range of detection
    4. Km of substrate(s) (for enzyme assays)
    5. Stability of reagents
    6. Stability of quenched assays or fixed and stained cells
    7. Intra-day variability (e.g., three plates run at different times in a single day)
    8. Inter-day variability (e.g., three plates run on three different days)
    9. Cell-seeding densities
    10. Other parameters particular to each individual screen

2. Miniaturization and Automation: If the assay has not already been optimized for 384-well plates, evaluate suitability of miniaturization. Miniaturization is highly encouraged as it can drastically reduce the amount of reagents, consumables, and compound used in the screen. Determine which steps will be performed on the robot, with a bulk dispenser, with a plate washer, or by hand with multichannel pipettors.

3. Initial assay statistics are determined, using appropriate positive and negative controls, on the same instrument that is to be used for screening. Important assay statistics include Signal/Background (S/B), Signal to Noise Ratio (SN) and Z’-Factor. There is no absolute cutoff criterion for either S/B or SN, but at least one of these values needs to be reproducibly high in order to arrive at an acceptable Z’-Factor of >0.5. Bear in mind that the higher the Z’-factor (up to a value approaching 1.0), the more reliable the initial hit-calling is likely to be. Initial false-positive rate and false-negative rate predictions should be possible after this step. 

4. Perform a small validation screen in duplicate (n=2), preferably run on two (or more) different days. Either the NIH Clinical Collection (6x96-well plates) or one of the DTP (NCI) sets (up to 10x96-well plates) is recommended for the validation library. In addition to being excellent tools for the validation of an assay for screening, these sets can provide novel pharmacological insight into the target of interest. 

5. Analyze the validation screen on a correlation plot. At this point one should have a good idea of the reproducibility of the screen and the false positive/negative rate. A decision is made whether the assay is ready to go on to the next step. An SOP should be written prior to initiation of the screening phase.


Step Two: Screening

1. A schedule and timeline for the screening campaign are created. Screening time can vary significantly depending on the assay format and number of compounds in the screen. Very roughly, plan for 2-4 weeks for a biochemical screen and 4-12 weeks for a cell-based screen. Default screening concentration for compounds is 10uM, which will equate to 0.5% DMSO if screening exclusively from a 2mM stock library.

2. Dataset from the screen is merged, processed and analyzed. Final hit-calling criteria are determined and a hit list is drawn up.  


Step Three: Reconfirmation and Follow-Up

1. Hits are cherrypicked from a cherrypicking copy of the compound library. Reconfirmation is typically done in triplicate at the screening concentration.

2. Reconfirmed hits can be re-ordered in larger quantities at this point, or synthesized in-house if feasible. Usually, higher quantities of compound than are present in the screening library are necessary to make full dose-response source plates that can be used for IC50 determinations in the primary assay as well as secondary and counter assays.