The cell proliferation guide

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.

Overview

Identifying proliferating cells
DNA synthesis
Cellular metabolism
Proliferation proteins
Scoring cell proliferation

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) Anti-BrdU antibody - Proliferation Marker (ab1893)
	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	Idoxuridine (ab142581) Anti-BrdU antibody [BU1/75 (ICR1)] (ab6326)
	EdU	Immunoassay to quantify cells in G1, S, and G2/M Trace cell cycle kinetics 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	Thiazolyl blue tetrazolium bromide (MTT) (ab146345)
	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	XTT sodium salt (ab146310)
	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	WST-1 Cell Proliferation Reagent (ready to use) (ab155902)
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	Anti-PCNA antibody [PC10] (ab29)
	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	Anti-Ki67 antibody (ab15580) Anti-Ki67 antibody [SP6] (ab16667)
	MCM-2	Immunoassay to detect cells in G1, S, G2 and M Prognostic and diagnostic value in some cancers	Scoring is subjective	Anti-MCM2 antibody (ab4461) Anti-MCM2 antibody [EPR4120] (ab108935)

DNA synthesis

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 cycle^1,2.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BrdU antibody - Proliferation Marker (ab1893)

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections of Ramos cell line xenograft tissue sections using an anti-BrdU antibody (ab1893).

5-bromo-2'-deoxyuridine (BrdU) is a thymidine analog that is incorporated into newly synthesized DNA
Labels proliferating and daughter cells
Detected by staining with anti-BrdU antibodies
Can be used to accurately quantify the percentage of cells in G1, S, and G2/M, and trace cell cycle kinetics
Requires DNA denaturation (DNase, heat, or acid) to allow antibody access to BrdU
This disrupts DNA morphology and can destroy recognition antigens, impairing subsequent co-staining procedures
Discover how to set-up a BrdU assay to stain proliferating cells in vitro or in vivo using BrdU with our protocol

IdU and CldU

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IdU [2F8] antibody (ab187742)

Immunohistochemical analysis of paraffin-embedded colon tissue from IdU injected mouse, labeling IdU with an anti-IdU [2F8] antibody (ab187742).

5-Iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU) are both thymidine analogs that are incorporated into newly synthesized DNA
Label proliferating and daughter cells
Ideal for time course studies
Can be used to study DNA replication fork progression rates, stability, or origin firing by sequential labeling with CldU and IdU.
Detected by staining with anti-BrdU or anti-IdU antibodies
NB some anti-BrdU antibodies cross-react with CldU (but not IdU) and some with IdU (but not CldU). These should not be used in conjunction with BrdU.
Requires DNA denaturation (DNase, heat, or acid) to allow antibody access to BrdU
This disrupts DNA morphology and can destroy recognition antigens, impairing subsequent co-staining procedures

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.

5-ethynyl-2′-deoxyuridine (EdU) is a thymidine analog that is incorporated into newly synthesized DNA
Labels proliferating and daughter cells
Can be used to accurately quantify the percentage of cells in G1, S, and G2/M
Unlike BrdU, IdU, and CldU, EdU detection uses ‘click’ chemistry rather than the addition of a detection antibody
EdU’s ethynyl group covalently crosslinks with a fluorescent azide (eg an Alexa Fluor^®), which is small enough to diffuse freely through native tissues and DNA
DNA does not need to be denatured, meaning EdU can be used in subsequent co-staining experiments
Simplified protocol due to lack of antibody and denaturation steps

Related products

MTS Cell Proliferation Assay Kit (Colorimetric)

Proliferation proteins

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 origins^3–6. 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).

Proliferating cell nuclear antigen (PCNA) is expressed mainly in late G1 and S, to a lesser extent in S and G2, and is low or absent in G0 and early G1
Widely used general cell proliferation marker⁹
Reported prognostic significance in certain cancers
Results relate only the number of proliferating cells, not the rate of proliferation

Ki67

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded sections from mouse spleen, labeled using an anti-Ki67 antibody (ab15580).

Ki67 nuclear antigen is expressed in the cell cycle phases G1, S, G2 and M, but is absent in G0
Ki67 index is widely used as a tumor marker in research and pathology
Prognostic and diagnostic value in many cancers⁹
Ki67 index correlates with the course of neoplastic disease and can be used to assess patient survival and cancer progression
Results relate only the number of proliferating cells, not the rate of proliferation
Often more specific than PCNA⁶

MCM-2

MTT
	2-(4,5-Dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) MTT is soluble in water Respiring cells convert MTT to a purple formazan dye Resulting dye is insoluble in water Primarily an endpoint measurement due to needing to dissolve the dye crystals in a solvent

XTT
	2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) XTT is soluble in water Respiring cells convert the XTT to an orange colored formazan dye Resulting dye is soluble in water No solubilization required prior to quantification Sensitivity equal to or better than that of MTT

WST-1
	Water-soluble tetrazolium salt-1 (WST-1) Respiring cells convert WST-1 to a dye that is measured at OD420–450 Resulting dye is soluble in water More sensitive than MTT, XTT or MTX Assay can be performed in the sample microtiter plate No additional steps like washing harvesting or solubilization