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The cell proliferation guide

Related

  • Cell biology resources
    • Cell cycle regulation
      • Cell cycle analysis protocol
        • Cell proliferation and cell cycle assays guide
          • Primary antibody resources
            • Antibody guide
              • Ki67 in focus
                • Ki67: a comparison of leading antibodies
                  • Ki67 resources
                    • Cell imaging
                      • Direct vs indirect immunofluorescence
                        • ICC protocol
                          • Compare Alexa Fluor®488 to FITC
                            • Subcellular and cell membrane markers

                              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 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
                              •  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

                              • BrdU Cell Proliferation ELISA Kit (colorimetric) (ab126556)
                              • BrdU Cell Proliferation ELISA Kit (chemiluminescent) (ab126572)
                              • BrdU Immunohistochemistry Kit (ab125306)
                              • EdU Proliferation Kit (iFluor 488) (ab219801)


                              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

                              • 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 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