The Molecular Screening Facility provides a range of specialized services critical to high-throughput screening biology.  A critical aspect of the Facility’s services is an intensive consultation between prospective users and the facility’s managing and scientific directors. These interactions occur throughout the duration of a project, from target justification to selection and optimization of the most appropriate assay technologies, to data analysis and interpretation of high-throughput screens, and finally, post-screening advancement.

Library Management
Assay Development
High-throughput Screening
Post-Screening Studies
Data Handling and Analysis

Specialized Services

Library management

Laboratory staff is responsible for the collection, handling, and formatting of libraries for screening applications.  In addition, the laboratory staff handles all library transfer procedures, including pinning compounds to assay plates and “hit” picking.

Assay development

Guidelines to developing a miniaturized bioassay for High-throughput screening
Plate type
Assay volume
Optimization of assay variables
Quantitative assay evaluation

The overarching goal for Assay Development focuses on the timely delivery of robust and well validated biological assays ready for implementation in screening experiments. Users are encouraged to contact laboratory staff as early as possible and remain actively engaged throughout the entire process.  The laboratory staff provide:

     - Expertise in cell biology, molecular biology, enzymology, biochemistry, pharmacology, and microbiology.

     - Facilities for propagation of bacteria, yeast, insect cells, mammalian cells, and viruses (i.e. baculovirus, retrovirus).

     - Expertise in expression and purification of recombinant proteins.

     - Equipment and expertise for maintenance, quality control and molecular characterization of normal and engineered cells for cell-based assays under standard biosafety containment up to and including BSL-2.

     - Experience with a variety of biochemical, cellular, immunological, and image based assays using an array of equipment compatible with bench scale or HTS scale experiments.

Guidelines to developing a miniaturized bioassay for High-throughput screening

The process of assay development includes the identification of assay types for screening and determination of structure-activity-relationships, development of assay reagents, optimization of assay parameters for signal intensity, signal window, and precision, adaptation to automation, scalability, and quantitative assessment of the assay's fitness for screening. Assay parameters to optimize assay's include sensitivity to enable identification of compounds with low-potentcy, precision of biological response between wells and plates, accuracy of positive and negative control compounds with known pharmacology towards the intended target, and economic feasibility.

Plate type

Depending on the method of detection used by an assay, different types of plates are recommended. For a list of plates and suppliers, please consult with the screening laboratory staff. 

Assay volume 

For high-throughput screening, a biochemical or cell-based assay must be adapted to a microtiter plate format.  Assays are most commonly performed in 96-, 384-, or 1536-well assay plates.  This laboratory recommends that assays be miniaturized to a 384-well plate.  Assay volumes in 384-well plates range from 5 µl (in low-volume plates) to 100 µl (in standard plates). Due to inaccuracies in small-volume pipetting and the potential risk of spillage and/or cross-contamination at the high end, it is recommended that investigators use an assay volume of 30-50 µl in standard plates.  

Optimization of assay variables

Reagent stability:  It is important to test the stability of assay reagents for standard storage and handling conditions in order to define procedures that minimize the loss of activity.  In addition, it is essential to determine the effects of modifications in preparation procedures and lot variation on assay signal and variation.  These data are usually determined by subjecting the handling of one assay reagent to different conditions for various times prior to addition to the assay.

Reaction stability:  It will be essential to determine the stability of the assay’s signal window and precision with respect to incubation time of various steps in the assay protocol.  These data are expected to provide information that greatly aid in defining logistical questions related to screening and tolerance of the assay to potential delays encountered during screening experiments. 

DMSO sensitivity:  Small molecule libraries are commonly formatted at fixed concentrations in 100% DMSO.  Therefore, it is important to determine the compatibility of the assay’s response to a range of DMSO concentrations.  It is recommended that these studies are done early in the development process, as replicate plate experiments to assess an assay’s fitness for screening should be done in the presence of the anticipated final DMSO concentration.

Quantitative assay evaluation 

The Z´-factor calculation provides a widely accepted method to quantitatively assess the fitness of specific assay conditions (Zhang et. al. 1999).  The Z’-factor is a unit-less numeric value that takes into account both the signal window and precision of measuring the maximum and minimum control signals in replicate wells.  Newly developed assay conditions are considered validated for high-throughput screening after a replicate plate experiment has demonstrated the assay conditions meet minimum criteria for acceptance. Each experiment should be performed on at least 2 full 384 well plates where 1⁄2 of the wells contain minimum signal controls and 1⁄2 of the wells contain maximum control signals, set up on 3 independent days starting from scratch. For previously established/tested assays, a single day replicate plate experiment will be sufficient. This assessment will produce a statistically significant data set for evaluation. 

To quantitatively rank assay conditions, calculate Z´ from the data collected, using:


SD + = positive control standard deviation

SD - = negative control standard deviation

Ave + = positive control average

Ave - = negative control average

Self Calculating Z-factor MS Excel workbook.

The following table is useful for evaluating the potential performance you might expect from these defined assay conditions during screening experiments.

1 > Z´ > 0.9 An excellent assay

0.9 > Z´ > 0.7 A good assay

0.7 > Z´ > 0.5 Hit selection will benefit significantly from any improvement

0.5 = Z´ The absolute minimum recommend for high throughput screening

This table represents general guidelines that are widely accepted in the academic screening community. If optimization is needed, different assay conditions should be compared and ranked by their Z´-factor values until suitable conditions are found.  Please note that screening results rarely achieve the high quality levels seen during the pilot phase using defined controls.

Ji-Hu Zhang, Thomas D. Y. Chung and Kevin R. Oldenburg (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen 4:67-73.

For a very thorough discussion of guidelines to developing specific assay types for HTS please see



Pilot screening 

After establishing an optimized assay protocol that meets acceptable criteria in a replicate plate test, a pilot screen of 2,000 to 3,000 compounds from a reference library with a high-degree of pharmacophore content is recommended.  The purpose of the pilot screen is to assess the behavior of the assay conditions and automation procedures you have optimized during assay development under actual screening conditions.  In addition, this pilot screen will be used to establish criteria for ‘hit’ conditions and selection.

Production screens 

The facility has the capability to process upto ~100 microtiter plates per day depending on the assay type and number of steps. Facility staff will work with investigators/users to determine screen logistics (time considerations, libraries, amounts/volumes of reagents, etc). For newly developed assays, it is recommended that the assay conditions be tested in a pilot-scale screen (see above).  For previously developed assays, demonstration of acceptable criteria in a single day replicate plate procedure will be sufficient to proceed with a production screen.  When performing a production screen, it is preferable to process as many plates as possible during usage of the equipment and laboratory staff time.

To schedule any screen, contact Joseph Salvino.  Do not make arrangements with staff of the facility.  Please plan to schedule your screen well in advance of the date you would like to start your experiment to eliminate scheduling conflicts and to ensure that the libraries are properly formatted.

Only screening facility personnel are permitted to handle library stock plates, and thus they perform all compound transfers from library plates into assay plates. Screening facility personnel will not be responsible for conducting any other parts of a screen unless agreed upon prior to the initiation of the screen, and will be charged as assisted use.

The laboratory conducting the screen is responsible for purchasing all plasticware to be used for the screen, this includes pipette tips, plates, and any other materials specific to your screen (reagent troughs, plate seals, tissue culture reagents, assay reagents, labels for barcoding etc.). The screening laboratory has also negotiated some bulk discounting on plates and pipette tips for the liquid handling unit, and therefore, investigators can purchase these items from the screening facility at cost.

Post-screening studies

The laboratory staff will assist users with cherry-picking requests of candidate ‘hits’ from primary screens, preparation of plates with serial dilutions for IC50/Ki measurements, and development of orthogonal assays and counter-screens to confirm the accuracy and rank order potency of identified compounds. 

Data handling and analysis

In conjunction with the Wistar Cancer Center Bioinformatics core, infrastructure is being established with a broad range of capabilities for data handling and storage.  The goal is to provide the ability to analyze biological data sets from screening experiments and extract structure-activity relationship (SAR) information to assist prioritization of hit follow-up.  We anticipate these goals being achieved by implementing commercially available software applications and custom designed applications developed by staff in the Bioinformatics shared facility.

Planned capabilities:

 - Compound registration

 - Biological assay registration

 - Statistical analysis of biological assay data

 - Heterogeneous database querying and reporting

 - Clustering

 - Similarity-Diversity analyses

 - In Silico profiling

 - Structure-based docking and scoring

 - Designing and enumerating virtual libraries