What methods are used to measure cell proliferation?

Cell proliferation is one of the fundamental processes underlying organismal growth, development, reproduction, and heredity, and it is also a core step in cell fate regulation and the onset and progression of diseases. In in-vitro culture systems or at the tissue level, accurately evaluating cell growth rate and proliferative capacity is of great importance for studies on cell growth, differentiation, injury repair, and anti-tumor drug screening. From the perspective of detection principles, assays for cell proliferation can be broadly classified into five categories: metabolic activity–based assays, DNA synthesis–based assays, cell number–based assays, proliferation-associated antigen assays, and ATP content–based assays. Among these, methods based on changes in DNA content and on cellular metabolic activity are currently the most widely used and are the first-choice readouts for most pharmacological efficacy and toxicity experiments.

I. Metabolic activity assays

【Principle】

During proliferation, the activity of dehydrogenases in cellular organelles such as mitochondria increases. After adding tetrazolium salt substrates (e.g., MTT, WST-8) to cells, metabolically active viable cells reduce them to colored formazan or formazan salts, and within a certain range the amount of product formed is proportional to the number of viable cells. Measuring the absorbance of the culture system with a microplate reader can thus indirectly reflect cellular metabolic level and proliferative status.

【Representative methods or reagents】

Typical metabolic activity assays include the MTT assay and the CCK-8 (WST-8) assay. In the MTT assay, MTT is reduced by succinate dehydrogenase and cytochrome c in the mitochondrial respiratory chain to form insoluble blue-purple formazan crystals, which are then dissolved in an organic solvent such as DMSO; absorbance at 490 nm is measured to estimate viable cell number. The CCK-8 assay is based on the reduction of WST-8 by mitochondrial dehydrogenases in the presence of the electron mediator 1-Methoxy PMS to generate a water-soluble orange formazan dye. The product dissolves directly in the medium without an additional dissolution step, and its absorbance is proportional to viable cell number; therefore, CCK-8 is widely used as an upgraded alternative to MTT in proliferation and cytotoxicity assays.

【Advantages】

Simple operation, suitable for high-throughput formats such as 96-well plates; objective readout that facilitates dose–response curve analysis. In particular, CCK-8 offers high sensitivity, a wide linear range, and relatively low cytotoxicity.

【Limitations】

An indirect measurement that is susceptible to interference from metabolic status and drug-induced mitochondrial toxicity; the MTT assay is more labor-intensive and has moderate reproducibility, while CCK-8 is relatively more expensive.

【Applicable scenarios】

Suitable for experiments where trends and relative differences are the focus, such as cell proliferation, drug cytotoxicity, and sensitivity pre-screening; it is one of the routine first-choice readouts for most efficacy and toxicity studies.

II. DNA synthesis assays

【Principle】

DNA synthesis is the core event of the S phase of the cell cycle and one of the most direct molecular features of cell proliferation. By adding thymidine analogs (e.g., BrdU or EdU) to cells, these molecules are incorporated into newly synthesized DNA strands during replication. Subsequent detection with specific antibodies or click-chemistry fluorescent probes enables quantitative or semi-quantitative assessment of the proportion of S-phase cells and DNA synthesis level, thereby evaluating cell proliferation.

【Representative methods or reagents】

Common analogs include BrdU (5-bromo-2′-deoxyuridine) and EdU (5-ethynyl-2′-deoxyuridine). BrdU is a classical S-phase marker incorporated into nascent DNA and detected immunologically using anti-BrdU monoclonal antibodies. EdU utilizes its ethynyl group to undergo a copper-catalyzed click reaction with azide-labeled fluorescent dyes, allowing direct labeling without harsh DNA denaturation.

【Advantages】

Directly targets DNA replication with high specificity, enabling accurate evaluation of S-phase fraction and proliferative rate; suitable for quantitative and spatial analyses when combined with flow cytometry or microscopy. The EdU method avoids strong DNA denaturation, causes less disruption to cells and tissue architecture, is simpler and faster, and provides higher signal-to-noise, making it especially suitable for proliferation analysis in tissue sections, organoids, and whole organs.

【Limitations】

BrdU assays are more complex, require acid/heat/enzymatic denaturation, and must be protected from light; EdU is relatively expensive, and long-term or high-dose treatment may have potential effects on the cell cycle.

【Applicable scenarios】

Preferred for cell-cycle studies, mechanistic research on anti-proliferative drugs, and proliferation localization analysis at tissue or organ level; the first choice when precise S-phase assessment is needed.

III. Viable cell counting assays (Trypan Blue, etc.)

【Principle】

Viable cells maintain intact membranes with selective permeability and are not readily penetrated by large dyes; dead or severely damaged cells have increased membrane permeability, allowing dyes to enter and bind cellular components, producing visible coloration. Trypan Blue is a typical exclusion dye: it does not enter viable cells within a short time but quickly enters dead cells with compromised membranes, staining them blue. By counting total cells and stained cells under a microscope, cell viability can be estimated.

【Representative methods or reagents】

The classic method is Trypan Blue staining and counting. After brief incubation of cells with dye, unstained (viable) and blue (dead) cells are distinguished on a hemocytometer and the proportion calculated.

【Advantages】

Intuitive principle, simple operation, and minimal equipment requirements—only a standard microscope and counting chamber are needed. It serves as an “on-site quick check” during cell culture and is extremely low-cost, enabling repeated use.

【Limitations】

Only roughly distinguishes live/dead cells and is insensitive to mild damage or vitality differences; prolonged staining can yield false positives, and results are relatively subjective with higher operator-dependent error.

【Applicable scenarios】

Used for routine “health checks” during culture (e.g., before passaging, before modeling, after thawing) to pre-evaluate cell quality, rather than as a rigorous endpoint proliferation quantification method.

IV. Proliferation marker assays (Ki-67, PCNA, MCM, etc.)

【Principle】

Certain nuclear proteins are highly expressed only during the proliferative phases of the cell cycle (G₁, S, G₂, M) and are nearly absent in quiescent (G₀) cells. Detecting these proliferation-related markers can indirectly indicate whether cells are proliferating and estimate the proportion of proliferative cells in a population, thereby assessing proliferative activity in tissues or cell cohorts.

【Representative methods or reagents】

Common markers include Ki-67, PCNA, and MCM proteins. Their expression levels and localization are evaluated by IHC, IF, or flow cytometry to assess tissue or population proliferative activity.

【Advantages】

Allows in situ detection in tissue sections or organoids, providing both quantitative and spatial information; markers such as Ki-67 have established applications in tumor grading and prognosis.

【Limitations】

Reflects the “fraction of cells in the proliferative cycle,” which is not equivalent to absolute changes in cell number; staining outcomes are strongly influenced by antibodies, antigen retrieval, and reader experience, requiring strict quality control.

【Applicable scenarios】

Suitable for assessing proliferation indices and regional distribution in pathological sections, organoids, and in vivo models; commonly used in tumor biomarker analysis and long-term pharmacodynamic studies.

V. ATP level assays

【Principle】

ATP is the most direct cellular energy “currency,” supporting nearly all metabolic and biosynthetic activities. Viable cells contain stable ATP levels, whereas ATP in dead or severely damaged cells rapidly degrades or is depleted. Within a certain range, total ATP content correlates linearly with viable cell number. A common assay is based on the bioluminescent reaction of firefly luciferase with luciferin: in the presence of ATP, luciferase catalyzes luciferin oxidation and emits photons; luminescence intensity is proportional to ATP concentration and can be rapidly read with a luminometric plate reader.

【Representative methods or reagents】

Typically performed using ATP bioluminescence assay kits. Cells are lysed and reacted with the luciferase/luciferin system, and luminescence is measured with a luminometer. Some systems use dual-luciferase assays for internal normalization.

【Advantages】

Extremely high sensitivity and wide linear range, enabling detection of low cell numbers or subtle ATP changes; rapid and objective readout, well suited for high-throughput screening and energy-metabolism studies.

【Limitations】

Requires instruments capable of luminescence detection and is sensitive to temperature, timing, and reagent condition. ATP levels are influenced not only by cell number but also by metabolic state, so interpretation should be combined with other indicators.

【Applicable scenarios】

Applicable to large-scale drug screening, cytotoxicity evaluation, and energy-metabolism research. Often used together with metabolic activity and DNA synthesis assays to complement and validate proliferation results from an energy perspective.

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