The cell proliferation guide
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All of the tools and techniques you need to stain and score cell proliferation.
Cell proliferation can be used to assess normal cell health, to measure responses to toxic insult, or as a prognostic and diagnostic tool in several cancers. The available markers typically look at DNA levels or synthesis, cellular metabolism, or proliferation-specific proteins.
This guide highlights the most common methods to mark and score cell proliferation.
- BrdU
- IdU and CldU
- EdU
- MTT
- XTT
- WST-1
- PCNA
- Ki67
- MCM-2
Identifying proliferating cells
Below are some of the best methods used to study cell proliferation. We’ve highlighted in green our recommended techniques for each method type.
For investigating cell proliferation in fixed samples, we suggest using Ki67 because it is well-established and highly-cited across both the basic and clinical research areas. MCM-2, another proliferation marker, is steadily gathering data around its use a prognostic marker in certain cancers, making this something to pay attention to as the research continues. For live cells, EdU is the preferred choice.
Method | Marker | Use and benefits | Limitations | Products |
DNA synthesis | BrdU | Immunoassay to quantify cells in G1, S, and G2/M Trace cell cycle kinetics | Requires DNA denaturation, impairing co-staining and disrupting DNA morphology Complex protocol | BrdU (5-bromo-2'-deoxyuridine) (ab142567) Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326) |
IdU & CldU | Immunoassay to study DNA replication fork progression rates, stability or origin firing Two dyes (against IdU and CldU) allow more complex experiments than with a single dye | Requires DNA denaturation, impairing co-staining and disrupting DNA morphology Complex protocol | Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326) | |
EdU | Immunoassay to quantify cells in G1, S, and G2/M
Simple protocol, without DNA denaturation | Can be expensive | 5-Ethynyl-2'-deoxyuridine (5-EdU) (ab146186) EdU Proliferation Kit (iFluor 488) (ab219801) | |
Cellular metabolism | MTT | Biochemical assay to indirectly quantify proliferating (respiring) cells Simple method | Toxic to cells Insoluble in water – needs to be dissolved in a solvent. Endpoint measure only Metabolic assays may not accurately represent changes in cell growth | |
XTT | Biochemical assay to indirectly quantify proliferating (respiring) cells Simple method More sensitive than MTT | Sensitivity varies Metabolic assays may not accurately represent changes in cell growth | ||
WST-1 | Biochemical assay to indirectly quantify proliferating (respiring) cells Simple method More sensitive than MTT and XTT | Metabolic assays may not accurately represent changes in cell growth | ||
Proliferation proteins | PCNA | Immunoassay to detect cells mainly in late G1 and S phases Prognostic value in some cancers | Scoring is subjective Can be less sensitive and specific than Ki67 methods | |
Ki67 | Immunoassay to detect cells in G1, S, G2 and M Prognostic and diagnostic value in some cancers Huge body of supporting evidence | Scoring is subjective Can be less sensitive and specific than MCM-2 in some cancers | ||
MCM-2 | Immunoassay to detect cells in G1, S, G2 and M Prognostic and diagnostic value in some cancers | Scoring is subjective |
The most reliable and accurate method of assessing cell proliferation is a measurement of DNA-synthesizing cells. This relies on incubating live cells with compounds capable of being incorporated into newly synthesized DNA. These compounds can then be detected with a reporter.
Thymidine analogs are the compound of choice to be incorporated into DNA, substituting thymidine during DNA replication. However, it is important to be aware that these thymidine analogs can lead to mutations and DNA damage in some instances and thereby affect the cycle cycle1,2.
This method is suitable for immunohistochemistry (IHC), immunocytochemistry (ICC), ELISAs, flow cytometry, and some multiplex assays. Combining IdU and CldU allows for time course studies when studying DNA replication by sequential labeling.
- Accurate and reliable
- High- and low-throughput options
- Protocol can be lengthy
- DNA denaturation prohibits subsequent co-staining experiments (not a concern with EdU)
| BrdU | |
Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections of Ramos cell line xenograft tissue sections using an anti-BrdU antibody (ab1893). |
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| IdU and CldU | |
Immunohistochemical analysis of paraffin-embedded colon tissue from IdU injected mouse, labeling IdU with an anti-IdU [2F8] antibody (ab187742). |
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![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IdU [2F8] antibody (ab187742)](https://a.abcam.com/ps/products/187/ab187742/Images/ab187742-220178-ab187742IHCb.jpg)
| EdU | |
BrdU assays (left) needs the DNA to be denatured in orderto allow an anti-BrdU primary antibody access to the BrdU molecule. EdU assays (right) rely on 'click' chemistry, in which the fluorescent azide can freely bind the EdU molecule. |
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Related products
- BrdU Cell Proliferation ELISA Kit (colorimetric) (ab126556)
- BrdU Cell Proliferation ELISA Kit (chemiluminescent) (ab126572)
- BrdU Immunohistochemistry Kit (ab125306)
- EdU Proliferation Kit (iFluor 488) (ab219801)
Rather than looking at DNA synthesis, it is possible to assay cell proliferation by measuring the metabolic activity of your cells in culture via tetrazolium salts. These salts form a dye when present in a metabolically active environment. The resulting color change of the media can be quantified in a spectrophotometer, giving an indication of the extent of proliferation.
Although sensitive, some of these salts are insoluble in normal culture medium, and the dye crystals often need to be dissolved in a solvent like DMSO or isopropanol. However, others are soluble in culture medium and nontoxic.
- Accurate to varying degrees
- High- and low-throughput options
- Protocol is simple
- Some dyes require toxic solvents
- Metabolic assays may not accurately represent changes in cell growth
| MTT | |
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| XTT | |
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| WST-1 | |
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Related products
Another method to study cell proliferation is by looking at specific proteins that are expressed in proliferating cells, but absent from non-proliferating cells. This requires the use of specific primary antibodies against the antigens expressed during proliferation.
These antigens are typically expressed in the perinuclear or nuclear interior regions across all cell cycle phases except G0, making them excellent cellular markers for proliferation. Ki67 is a very popular proliferation marker and is routinely used in pathology labs due to its diagnostic and prognostic power in cancer. PCNA is another common marker, yet multiple studies have shown that Ki67 is more sensitive and specific when evaluating cell proliferation in tumors from various origins3–6. A maker growing in prominence is MCM-2, and recent work suggests this may be a better choice for informing cancer prognoses than Ki67 and PCNA7,8.
However, much of the data is inconclusive regarding a ‘best’ maker of proliferation, especially in a clinical context.
These immunoassays are excellent for fixed tissue samples and analysis by IHC.
- Accurate and reliable
- Large body of supporting data
- Clinical diagnostic and prognostic value in some cases
- Limited high-throughput options
- Scoring of results can be subjective
- Conflicting data around the ‘best’ marker of cell proliferation in a clinical setting
| PCNA | |
Immunohistochemical analysis of frozen sections from adult zebrafish intestine, labeled with an anti-PCNA antibody [PC10] (ab29). |
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| Ki67 | |
Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections from mouse spleen, labeled using an anti-Ki67 antibody (ab15580). |
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| MCM-2 | |