Innovating Your Drug Discovery

Do-It-Yourself: Benefits and Challenges of Creating Your Own Cell-Based Assays

Do-It-Yourself Cell-Based Assays

Cell-Based AssaysCell-based assays are useful for understanding cellular pathways; characterizing targets and their function; identifying, screening, and optimizing ligands or drugs in their cellular environment; and more. These assays are typically purchased through cell-based assay providers as a product or service for the convenience of saving time related to assay conceptualization, development, optimization, and validation.  

What if the assay you are looking for doesn’t exist?  You may engage a company to develop a custom assay or develop your own cell-based assay in-house – the do-it-yourself (DIY) option. Whichever is the case, DiscoverX can help you.

DiscoverX has a toolbox of cells and reagents which can jumpstart and accelerate your DIY cell-based assay approach. The DIY cell-based approach is most advantageous when there is a need for developing an assay that fits your specific project needs. For example, your assay may involve analyzing different cell types or modifications of your protein target (e.g. looking at mutants or orthologs). Although some molecular biology and cellular assay development expertise, time commitment, and appropriate laboratory tools are required, the opportunity for having an assay that meets your exact needs is worth the investment.

Benefits and Challenges

The ultimate benefit of creating your own custom, cell-based assay is obtaining an ideal assay for your application. Another benefit is related to the effort required for assay development. This effort is not only rewarding to your organization, but also to the scientists who gain an overwhelming amount of expertise that advances their skill set and benefits their work experience. Building your own assay can lead to a more intimate understanding of the assay principle that otherwise might not occur, which can open doors for consideration of the assay for multiple applications.

The challenges of DIY cell-based assays are related to the assay developers and the time required to develop, optimize, and validate the assay to ensure success. To alleviate these challenges, consider the following:

  • Have the right expertise to save time, ensure the highest likelihood of assay success, interpret the results (during data analysis) effectively, and limit assay pitfalls.
  • Anticipate and understand the assay factors that may deteriorate assay performance, decrease cell viability, or cause inconsistent and irreproducible results. These factors include microplate plate edge-effects; pipetting errors; cell density variations; contaminators; temperature, timing, and CO2 variabilities; and instrumentation and detection parameters.
  • Choose the right cell type for your application. Consider immortal cell lines (e.g. CHO-K1, HEK 293, U2OS) or primary cells (e.g. HUVECs, hepatocytes).
  • Ensure you have the appropriate assay controls. Positive and negative controls indicate if the assay is functioning properly and provide a reference point for data analysis.
  • Consider the assay versatility for multiplexing, high-throughput, miniaturization, mix-and-read convenience, and use for multiple applications.

Ultimately, the decision to pursue a DIY cell-based assay project is up to each lab and project team, based on the time, resources, and expertise available.

Workflow and Tools Example

The workflow and tools needed to create your custom assay can vary depending on the design of the cell-based assay.  Below is an example of the creation of a functional, cell-based assay that is based on the enzyme fragment complementation (EFC) from DiscoverX.

The EFC technology is based on two recombinant β-galactosidase (β-gal) fragments – a large protein fragment (enzyme acceptor, EA) and a small peptide fragment (enzyme donor, ED), which are added as tags onto proteins in your assay. Separately, the β-gal fragments are inactive, but when combined, they form an active β-gal enzyme that hydrolyzes a substrate (detection reagent) to produce a chemiluminescent signal. This simplistic system allows for interrogation of biomolecular interactions for characterizing targets and pathways as well as advancing therapeutic drug screening and development programs.

When creating your own EFC cell-based assay, you will need the following tools:

  1. Cell culture and transfection reagents including tissue culture plates, media, and a facility appointed with proper tissue culture equipment and instrumentation (e.g. a standard luminometer).
  2. A parental cell line containing a reporter protein tagged with EA. The reporter protein is related to the type of assay being performed. For example, when looking at internalization of a membrane receptor protein to the endosome, the reporter protein would be an endosomal protein. Alternatively, for a GPCR β-arrestin recruitment assay your reporter protein would be β-arrestin.
  3. A plasmid to introduce your target of interest to be tagged with ED. Your target could be a GPCR, kinase, ion channel, transporter, nuclear protein, or other protein of interest.
  4. EFC detection reagents that includes the enzyme substrate for producing the chemiluminescent signal.
Workflow for cell-based assays

Example workflow: Generate a PathHunter® EFC assay by first creating an ED-tagged target protein plasmid using a cloning vector followed by transfection of this plasmid into a PathHunter EA parental cell line of interest. Perform antibiotic selection, detect the level of expression of the two fusion proteins, and perform an EFC assay in the presence of a ligand or compound. The assay type can be a functional assay (e.g. analyzing GPCR β-arrestin recruitment), binding assay (e.g. evaluating target engagement), or translocation assay (e.g. detection movement of proteins to the membrane, endosome, or nucleus). See below for resources related to these types of EFC assays.

DIY Assay Resources & Select Publications

  • DiscoverX build your own assays tools
  • Cytotoxicity DIY assays using retroviruses
  • Protein translocation DIY assays for GPCRs, ion channels, kinases, and nuclear proteins, and related blog
  • SH2-protein recruitment DIY assays for receptor tyrosine kinases (RTKs), cytokine receptors, and cytosolic tyrosine kinases (CTKs), and related webinar
  • Target engagement (compound-protein binding assay) DIY assay and related poster and webinars
    • ‘Evaluating Drug-Target Engagement in Living Cells via Cellular Thermal Shift Assay’ poster (accepted publication in progress)
    • ‘Development of Three Orthogonal Assays Suitable for the Identification and Qualification of PIKfyve Inhibitors.’ abstract
    • ‘Utilisation of the InCELL Hunter™ Target Engagement Cell-Based Assay for Profiling of ULK1 Inhibitors’ abstract
  • GPCR β-arrestin recruitment DIY assays and related webinar
    • ‘Reassessment of the pharmacology of sphingosine-1-phosphate S1P(3) receptor ligands using the DiscoveRx PathHunter and Ca(2+) release functional assays.’ abstract
    • ‘Identification and characterization of a small molecule antagonist of human VPAC(2) receptor.’ abstract
    • ‘C5a-stimulated recruitment of beta-arrestin2 to the nonsignaling 7-transmembrane decoy receptor C5L2.’ abstract