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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.

DNA synthesis

  • BrdU
  • IdU and CldU
  • EdU

Cellular metabolism

  • MTT
  • XTT
  • WST-1

Proliferation proteins

  • PCNA
  • Ki67
  • MCM-2


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)

Anti-IdU antibody [32D8.D9] (ab181664)

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 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

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


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


Cellular metabolism

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
  • 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


Related products


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 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).

  • 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