In recent years, cell proliferation has been a hot topic in the field of biology. It is not only crucial for the normal growth and development of organisms but also underpins many pathological processes, such as cancer development and treatment response. Understanding how cells proliferate and how to accurately measure cell proliferation is of great importance for both scientific research and clinical applications. Today, we will provide a detailed introduction to the basic concepts of cell proliferation, commonly used detection methods, and the advantages and limitations of these methods.

The Importance of Cell Proliferation and Cell Proliferation Assays

Cell proliferation refers to the process by which cells divide under the regulation of cell cycle factors, involving complex reactions such as DNA replication, RNA transcription, and protein synthesis. Cell proliferation is fundamental to the growth, development, reproduction, and heredity of living organisms, making it a crucial characteristic of life.

Figure 1: Cell proliferation

Common Methods for Detecting Cell Proliferation

Detection of DNA Synthesis

Evaluating cell proliferation by directly measuring the synthesis of DNA is one of the most accurate methods. During cell proliferation, One of the most significant changes is DNA amplification, which can be used to assess changes in cell number by detecting variations in DNA quantity.

01 ³H-TdR

Also known as the radiolabeled nucleotide method, thymidine (TdR) is a base unique to DNA and is essential for DNA synthesis. Radioactive isotope ³H-labeled TdR acts as a precursor for new DNA synthesis. By measuring the radioactivity intensity of cells, cell proliferation can be inferred. However, due to its radioactive nature, this method is now rarely used.

02 BrdU (5-Bromo-2'-deoxyuridine) Incorporation Assay

BrdU is a thymidine analog that can replace thymidine and be incorporated into newly synthesized DNA. Labeled antibodies (e.g., with biotin or fluorochrome) can then detect BrdU in the DNA. By measuring this labeling, cell proliferation can be assessed.

03 EdU

An advanced alternative to BrdU, EdU is a thymidine analog that incorporates into newly synthesized DNA during replication. Using a click chemistry reaction, EdU molecules can bind to fluorescent or biotin probes, allowing new DNA to be labeled and subsequently detected to assess cell proliferation.

Figure 2. Principle of the EdU Assay Kit

Figure 3. HeLa cells treated with 10 μM EdU (E-Click EdU Cell Proliferation Imaging Detection Kit/E-CK-A377) for 2 hours. Fluorescence microscopy detection results, proliferating cells (red) and cells counterstained with DAPI (blue).

Fluorescent Labeling of Live Cells

01 CFSE

CFSE is a method widely used in recent years for detecting cell proliferation. CFDA SE is a cell-permeable fluorescent dye that is non-fluorescent in its native state. Once it enters live cells, intracellular esterases catalyze its conversion into carboxyfluorescein succinimidyl ester (CFSE), which emits a strong green fluorescence. The generated CFSE cannot cross the cell membrane and remains within the cell. It also spontaneously and irreversibly binds to intracellular amines, coupling to cellular proteins. This characteristic makes CFSE an important tool for tracking cell proliferation and fluorescent labeling.

Figure 4. Bone marrow-derived dendritic cells from mice induced in vitro and matured with LPS were co-cultured with normal mouse spleen cells (stained with CFSE (E-CK-A345)) for 72 hours. Subsequently, the cells were co-stained with Elab Fluor Red 780 Anti-Mouse CD8a (E-AB-F1104S) and APC Anti-Mouse CD4 (E-AB-F1097E), and T cell proliferation was analyzed using flow cytometry.

Detection of Cell Metabolic Activity

01 MTT 

MTT is a tetrazolium dye that can be reduced by certain dehydrogenases in mitochondria to form a dark purple crystalline product called formazan. In the presence of a specific solvent, formazan can be completely dissolved, and its absorbance is measured at around 570 nm using a spectrophotometer. The higher the cell proliferation, the higher the absorbance.

02 CCK-8

Cell Counting Kit-8 (CCK-8) is a rapid and highly sensitive assay based on WST-8, widely used for cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT which can be reduced to an orange-yellow formazan by some dehydrogenases in mitochondria in the presence of electron-coupling reagents.  The absorbance of formazan is measured at around 450 nm using a spectrophotometer. The higher the cell proliferation, the higher the absorbance.

Detection of Cell Proliferation-Related Antigens

Certain antigens are present only in proliferating cells and are absent in non-proliferating cells, making them markers of cell proliferation. The expression of these specific proteins, such as Proliferating Cell Nuclear Antigen (PCNA), Ki-67, and Minichromosome Maintenance proteins (MCM), can be detected using methods like Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF). These methods allow for the assessment of cell proliferation by identifying the presence and levels of these proliferation-specific antigens.

Comparison of Common Detection Methods

Elabscience® Cell Proliferation Detection Related Products

Assay Kits:

Antibodies: