What is Cell Proliferation?
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.
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 protocol flowchart
Factors to Consider When Choosing a Cell Proliferation Assay
1. Detection Method
Click on the common detection methods listed below to learn more.
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.
2. Sensitivity and Specificity
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 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.
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.
3. Compatibility with High-Throughput Screening (HTS)
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.
In Summary
| 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 |
Key Considerations in Multispan's Cell Proliferation Assays
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.
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.
1. Preservation of 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.
2. High-Throughput Screening (HTS) Compatibility
- 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
3. Rapid Turnaround and Customization
- One-month delivery for native cells, three months for engineered cells if single cell cloning is required.