Cell Proliferation Assays for Drug Discovery and High-Throughput Screening

Understanding Cell Proliferation in Drug Development

Cell proliferation refers to the process of cell division and reproduction. Understanding its biological mechanisms plays a crucial role in drug discovery research.

For example, in order to develop cancer therapeutics, researchers must gain insight into how tumor cells grow and spread. They must also be able to analyze and quantify how chemical, protein or RNA drugs affect the proliferation of these cells. Cell proliferation assays are essential tools in accomplishing these types of tasks.

Running a cell proliferation assay is a multi-step process, starting with seeding cells in well plates. These cells are treated with test compounds before being incubated. Using various detection methods, cell proliferation can then be measured.

Cell proliferation assay workflow

Common Cell Proliferation Assay Methods

Choosing the right detection method depends on the biological question, sensitivity needs, and compatibility with high-throughput systems. Below are widely used assay types:

Click on the common detection methods listed below to learn more.

ATP Luminescence
ATP assays measure cellular adenosine triphosphate levels. ATP serves as the primary cellular energy source for various cellular biological processes. Therefore its activity is used to determine the metabolic activity in cells. These assays rely on signals produced by ATP through catalyzing enzymatic reactions such as luciferases. The level of cell proliferation is quantified by the intensity of the signal readout, which can be luminescence, fluorescence or absorbance. Among the 3 different readouts, ATP Luminescence assays are highly sensitive, rapid with minimal incubation time, and well-suited for HTS.
DNA-binding dyes are commonly used to assess cell proliferation because the amount of DNA in the cellular samples correlates with cell growth and division. DNA binding dyes utilize fluorescent dyes (such as DAPI or Hoechst) to bind to DNA, and cell count is quantified corresponding to the intensity of fluorescence. DNA binding dyes are simple, requiring no complex reagents, and well suited for flow cytometry. They are also compatible with plate readers and fluorescent imaging systems.
Thymidine is actively incorporated into DNA during the S-phase of cell division, so are its analogs EdU (5-Ethynyl-2′-deoxyuridine) and BrdU (Bromodeoxyuridine). Both EdU and BrdU assays are highly specific to proliferating cells, and measure new DNA synthesis during replication. Measuring thymidine analogs is more specific to cell proliferation than measuring ATP while the EdU assay is the faster, more sensitive, less toxic and more HTS compatible option between the two analogs.
The XTT and MTT assays are both widely used to measure cell viability and proliferation by assessing metabolic activity, specifically mitochondrial dehydrogenase activity in the cells. In the MTT assay, mitochondrial dehydrogenases reduce the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to purple formazan crystals. Similarly, the XTT assay water-soluble 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) is converted into an orange-colored formazan product. Both converted products are quantified by absorbance. Both assays are commonly used in cytotoxicity and cell proliferation studies, with the XTT assay offering the advantage of a water-soluble product that eliminates the need for a solubilization step, making it more convenient for some applications.
Unlike MTT and XTT, the Alamar Blue assay is based on the reduction of resazurin (blue, non-fluorescent) to resorufin (pink, fluorescent) by metabolically active cells. resorufin can be measured by both absorbance and fluorescence. A key advantage of the Alamar Blue assay is that it is non-toxic, allowing for continuous monitoring of cell viability without requiring cell lysis, and it provides a reversible measurement, making it suitable for long-term or dynamic studies.
The Live/Dead Cell Viability Assays use dual-fluorescence staining to differentiate between live and dead cells based on membrane integrity. These assays typically employ membrane-penetrant Calcein-AM (green fluorescence, like FITC) to indicate live cells, which are metabolically active and have intact cell membranes, and non-membrane-penetrant Ethidium Homodimer-1 (EthD-1) (red fluorescence, like PE) to mark dead cells with compromised membranes. These assays provide a rapid, simple, and reliable method to assess cell viability, without requiring complex reagents or preparation steps. The readout is typically fluorescence, making them compatible with flow cytometry, fluorescence microscopy, and plate readers. They are widely used for applications where accurate and real-time viability assessment is essential, offering high sensitivity for detecting live and dead cells. However, they are less specific to proliferation since they primarily measure overall cell viability.

Assay Performance: Sensitivity, Specificity, and HTS Readiness

Sensitivity

Sensitivity refers to an assay’s ability to detect small changes in cell proliferation, making it particularly valuable for experiments involving low cell numbers, subtle signals, or compounds with minimal effects. In some cases, even the slightest variations in cell proliferation can be critical to measure. The sensitivity of an assay is largely determined by the detection method employed.

Specificity

Specificity refers to an assay’s ability to accurately measure cell proliferation without interference from other cellular processes and biological signals, like cell death, or non-target cells. A highly specific assay will be able to distinguish changes in cell proliferation from off-target effects or background noise, ensuring there are no false positives.

HTS Compatibility

To efficiently test a large number of compounds, the assay must be high-throughput screening (HTS)-friendly, enabling seamless integration with automated systems such as robotics and plate readers. It should be compatible with standard well plate formats (e.g., 96-, 384-, or 1536-well plates) to accommodate varying scales of experimentation. Additionally, the assay readout must be rapid, similar to the speed achieved with ATP luminescent assays or DNA-binding dye assays.

Comparison of Proliferation Assays

Assay Type Readout Sensitivity Specificity Assay Time HTS Compatibility Proliferation Assay Types of Reader Instruments
ATP Luminescence
Luminescence
High
High (directly linked to metabolic activity)
Short (15–60 min)
Excellent
Metabolic Activity-Based Assays
Plate readers
DNA Binding Dye
Fluorescence
Moderate to High
Moderate (based on DNA content, not cell cycle)
Moderate (30–120 min)
Good
Direct Cell Counting-Based Assays
Flow cytometers, Plate readers, Microscopes
EdU/BrdU Incorporation
Fluorescence
/Absorbance
High
High (specific to DNA replication)
Moderate (1–4 hrs)
Good
DNA Synthesis-Based Assays
Flow cytometers, Plate readers, Microscopes
XTT/MTT/Alamar Blue
Absorbance
/Fluorescence
Moderate to High
Low (linked to metabolism, not proliferation)
Moderate (1–4 hrs)
Excellent (XTT/Alamar Blue)
Metabolic Activity-Based Assays
Plate readers, Microscopes
Live/Dead Cell Viability
Fluorescence
High
Moderate (based on membrane integrity, not proliferation)
Rapid (30–60 min)
Good
Direct Cell Counting-Based Assays
Flow cytometers, Plate readers, Microscopes

Multispan’s Custom Cell Proliferation Assay Services

Multispan’s cell proliferation assay development and compound screening service provide researchers with more accurate, efficient, and physiologically relevant measurements and compound data for studying cell growth and drug responses.

Using primary cells or MULTISCREENTM stable cell lines, our assays are optimized for high signal-to-background ratios, HTS Z’-factors, and robust potency measurements.

Preserve Endogenous Signaling Pathways

  • We can measure in a panel of cell lines or primary cells derived directly from patients
  • Other phenotypic assays, second messenger assays, and more, designed for specific project needs
  • Diverse Readouts: XTT, EdU or Alamar Blue customized to reflect the target biology in flow cytometry, luminescence, TR-FRET, fluorescence, ELISA and other readouts.

High-Throughput Screening Scalability

  • One compound against many different cell lines or a millions compounds on one cell line
  • We have flexibility and scalability with a level of precision that’s uniquely available at Multispan

Rapid Delivery and Customization

  • One-month delivery for native cells, three months for engineered cells if single cell cloning is required.

FAQs

A cell proliferation assay measures the rate at which cells divide, offering vital insight into how cells respond to various treatments. This is especially important in cancer research, where understanding tumor growth and evaluating the impact of compounds on that growth is critical.

ATP luminescence assays are considered ideal for HTS applications. They offer high sensitivity and quick results, and integrate seamlessly with automated systems like robotics and multiwell plate readers. Their ability to detect subtle metabolic activity changes makes them effective for screening large numbers of compounds efficiently.

No — cell viability and proliferation are different. Viability determines whether a cell is alive, while proliferation tracks whether it’s actively dividing.

The XTT assay is more user-friendly than the MTT assay because its final product is water-soluble. This removes the solubilization step required in the MTT workflow, streamlining the process and reducing variability.

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