High-throughput screen (HTS) is a large-scale experiment to test the activity of collections of compounds against a biological target or pathway. The assays used for HTS of hit compounds are called primary screen. Once hits have been confirmed in the primary screen, assays need to be designed to eliminate false positives caused by the effect of compounds on the assay technology or format. Counter-screen is usually performed in parallel with or after the primary screen.

The reproducible interference of compounds on primary screen is a common cause of false positives. Counter-screen can be used to identify compounds that may interfere with the primary screen. The common types of compound interference include compound fluorescence, aggregation, luciferase inhibition, and redox reactivity.

In primary screen, a compound may be active in an assay but inactive toward the biological target. Counter-screens are often performed in the absence of the target to identify target-independent activity. For example, if the primary screen uses the firefly luciferase as a reporter to identify compounds that modulated biological targets, the assay used as a counter-screen would be a biochemical assay to identify compounds that inhibit firefly luciferase. In this case, compounds showing activity in the counter-screen should be eliminated from further analysis.

Counter-screen also aims at eliminating compounds with undesirable properties such as cytotoxicity which is a common source of false positives. The counter-screen used to measure compound cytotoxicity is frequently used for cell-based HTS and can be performed with or after the primary screen.

LOPAC hit validation and counter-screening Figure 1. LOPAC hit validation and counter-screening. (Wormald, M., et al., 2017)

Creative Biolabs provides counter-screen services for hit validation of drug discovery process thus to deal with data reproducibility crisis which is a big obstacle for drug research and development and may lead to failure and high risk of investment. We can drive forward and speed up your drug discovery process by eliminating the false positives and measuring the undesirable properties of multiple hit compounds.

Due to superior signal-to-noise ratios, bioluminescence-based assays are widely used in HTS. However, they are prone to generating false positives because of the inhibitory effect of compounds on luciferase enzyme. Creative Biolabs can offer counter-screen services based on our professional luciferase reporter assays with high-quality data, rapid turnaround time and reasonable price.

Taking advantages of state-of-the-art instruments and experienced scientists, Creative Biolabs is capable of designing and performing comprehensive cytotoxicity assays to obtain comprehensive information of hit compounds. We offer cell-based high-content cytotoxicity screening, a powerful time- and cost-effective approach which can provide multi-parametric information on cytotoxicity by simultaneously monitoring multiple toxicity markers such as cell number, cell viability, apoptosis, nuclear change (DNA content, nuclear size and morphology), plasma membrane permeability, intracellular calcium level, glutathione level, oxidative stress, mitochondrial membrane potential (MMP), and cytochrome c release. We are well-experienced in various cytotoxicity assays and are able to adjust the assays according to your needs.

In addition, Creative Biolabs has a dedicated assay development team that will work closely with you to design customized assays according to the primary screen of your drug discovery project. Our service will meet your specific needs fast at extremely competitive prices. If you need more information, please feel free to contact us at anytime. We look forward to working with you to help your drug research and development project succeed.


  1. Wormald, M., et al., 2017. Development of a homogenous high-throughput assay for inositol hexakisphosphate kinase 1 activity. PloS one, 12(11), e0188852.

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