Research Techniques Made Simple: Techniques to Assess Cell Proliferation

      Cell proliferation is commonly assayed in the laboratory for research purposes, but is increasingly used clinically to gauge tumor aggressiveness and potentially guide care. Therefore, both researchers and clinicians should have a basic understanding of techniques used to assess cell proliferation. Multiple cell proliferation assays exist, and the choice of method depends on the laboratory resources available, the types of cells/tissues to be studied, and the specific experimental goals. In this article, we identify four overarching categories of cell proliferation assays that signify various stages of the cell cycle: nucleoside-analog incorporation, cell cycle-associated protein detection, use of cytoplasmic proliferation dyes, and indirect measures of cell proliferation. Each method has strengths and limitations that should guide the dermatology investigator’s choice of assay.

      Benefits

      • Commonly utilized in laboratory research and increasingly in clinical practice.
      • Broad range of assays available to assess various stages of the cell cycle.
      • Some assays can be performed in conjunction with other stains, and some techniques maintain viable cells for follow-up experimentation.
      • Many methods are technically straightforward and utilize commonly available equipment.

      Limitations

      • Some assays require specialized equipment and/or materials.
      • Available sample(s) may be incompatible with certain techniques (eg, cytoplasmic dyes and thymidine incorporation cannot be used on formalin-fixed clinical skin specimens).
      • Some assays are lethal to cells, so further experimentation cannot be performed.

      Introduction

      Cell proliferation is defined as an increase in cell number secondary to cell growth and division (reviewed in
      • Schafer K.A.
      The cell cycle: a review.
      ). Assessing cell proliferation is a cornerstone of basic, translational, and clinical research and of clinical medicine. Given this pervasiveness, it is important for the clinical dermatologist and dermatology researcher alike to have a basic comprehension of cell proliferation and the assays most commonly used to measure it.
      A number of methods exist to measure cell proliferation, and they vary in regard to which phase of cellular growth and division they assay, the equipment and expertise required, whether additional studies can be performed in parallel or in series, and what types of cells/tissues can be studied by that assay. To simplify this topic, this article outlines the principal approaches used to assess cell proliferation based on three aspects of cell division: nucleoside-analog incorporation during DNA synthesis, cell cycle-associated proteins, and cytoplasmic proliferation dyes. A fourth section briefly reviews indirect methods of assessing cell proliferation via cell counting and viability and metabolic activity assays.

      The Cell Cycle

      Cellular proliferation results from progression through the cell cycle (Figure 1). The cell cycle has two major phases: interphase and mitosis (reviewed in
      • Schafer K.A.
      The cell cycle: a review.
      ). A cell spends most of its life in interphase, which is divided into three stages: Gap 1 (G1), Synthesis (S), and Gap 2 (G2). During interphase, the cell is growing and preparing for division. Cellular division occurs during mitosis, or (M) phase, which actually consists of both mitosis and cytokinesis. Mitosis refers to division of the nucleus resulting in equal separation of chromosomes and is subdivided into phases: prophase, prometaphase, metaphase, anaphase, and telophase. Cytokinesis is the equal division of the cell membrane, cytoplasm, and organelles. M phase results in two daughter cells identical to their parent cell. A fourth stage, Gap zero (G0), describes resting cells and cells that rarely or never divide.
      Figure 1
      Figure 1Schematic of the cell cycle depicting the phase detected by different proliferation assays. G0 is a relatively inactive state for the cell. G1 is a period of cell growth and preparation for DNA synthesis, or the S phase. Cell growth continues during G2 as the cell prepares for mitosis (M) phase. M phase consists of equal division of chromosomes and cytoplasmic components (termed cytokinesis) between two daughter cells. Cytoplasmic proliferation dyes are internalized during all phases of the cell cycle including G0, but proliferation is only appreciated once cytokinesis has occurred and the fluorescence intensity of the dye is halved in the daughter cells. Ki-67 is expressed during active phases of the cell cycle (G1-M). Nucleoside-analog incorporation assays (ie, [3H]TdR and BrdU) and proteins such as PCNA are specific to the S phase. BrdU, 5-bromo-2′-deoxyuridine; [3H]TdR, tritiated thymidine; PCNA, proliferating cell nuclear antigen.
      The stages of the cell cycle can be identified based on specific characteristics. For example, nucleosides are incorporated into replicating DNA exclusively during S phase, and histone protein H3 is only phosphorylated during M phase. Many proliferation assays take advantage of these unique characteristics of each cell cycle phase.

      Nucleoside-Analog Incorporation Assays

      During the S phase of the cell cycle, genome replication, DNA polymerases incorporate nucleosides (eg, deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine) into new strands of DNA. Nucleoside-analog incorporation assays introduce chemically or radioactively labeled nucleosides into the sample of interest, which are incorporated into newly synthesized DNA during S phase.
      A classic example of this strategy is the tritiated thymidine ([3H]TdR) incorporation assay whereby excess radiolabeled thymidine is added to cell cultures and allowed to incubate for multiple days. Excess [3H]TdR is then washed away and incorporated. [3H]TdR is measured using a liquid scintillation counter. The assay can be performed in vitro or ex vivo, but not in vivo. This method quantifies overall division compared with a control and is commonly regarded as reliable and accurate. Assays should be run in triplicate and using varying numbers of cells to generate a proliferation curve (Figure 2a). Drawbacks are that radioactive reagents must be handled and disposed of with caution, thymidine is measured by the scintillation counter per well of cells rather than per individual cell so the assay reveals nothing about an individual cell’s division history, and no additional assays can be performed with or after [3H]TdR incorporation (it is an endpoint assay) because the assay extracts DNA from cells that are then washed away in the process. A common use for [3H]TdR incorporation is to assess T-cell proliferation, for example, in response to therapeutic dendritic cells, as shown in Figure 2a (
      • Divito S.J.
      • Wang Z.
      • Shufesky W.J.
      • Liu Q.
      • Tkacheva O.A.
      • Montecalvo A.
      • et al.
      Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation.
      ).
      Figure 2
      Figure 2Example data from nucleoside- analog incorporation assays. (a) A tritiated thymidine ([3H]TdR) incorporation assay was performed to assess T-cell proliferation in response to treatment with therapeutic dendritic cells (DCs). Different ratios of irradiated dendritic cells (stimulator) (incapable of proliferating) to T cells (responder) were incubated together for 3 days; then [3H]thymidine was added to cultures and measured by liquid scintillation counter to generate proliferation curves shown. *** represents statistical significance between groups. Reprinted with permission from
      • Divito S.J.
      • Wang Z.
      • Shufesky W.J.
      • Liu Q.
      • Tkacheva O.A.
      • Montecalvo A.
      • et al.
      Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation.
      . (b) Fluorescent microscope images demonstrating increased cell proliferation (BrdU+, red) 4 days into wound healing in skin. Dapi images highlight the location of cell nuclei (purple).
      Reprinted with permission from
      • Yokoyama H.
      • Maruoka T.
      • Aruga A.
      • Amano T.
      • Ohgo S.
      • Shiroishi T.
      • et al.
      Prx-1 Expression in Xenopus laevis Scarless Skin-Wound Healing and Its Resemblance to Epimorphic Regeneration.
      .
      Another common nucleoside-analog incorporation assay employs 5-bromo-2′-deoxyuridine (BrdU), also a thymidine analog. Incorporated BrdU is detected by a BrdU-specific monoclonal antibody that may be bound directly to a fluorescent tag or measured indirectly via a secondary antibody. Fluorescence can then be measured via a flow cytometer or fluorescence microscopy (Figure 2b). Alternatively, chromogenic tagging (colored, rather than fluorescent) can be performed that requires only a standard light microscope for inspection. An added benefit of the assay is that DNA can be “captured” via the antibody (ie, bound by the antibody, then purified by immunoprecipitation, and subsequently sequenced). Further, BrdU can be used not only in vitro but also in vivo; it can be injected into an animal or added to the animal’s drinking water and ingested. BrdU is stably incorporated into DNA, so it persists for several months. Additional stains can be included in this assay, for example, to identify the particular type of cell that is proliferating. Lastly, there is no handling of radioactive material. Two negatives of this method are that it is an endpoint assay because staining requires cell membrane permeabilization and fixation, and it cannot identify cells that have undergone numerous rounds of division (reviewed in
      • Lyons A.B.
      • Blake S.J.
      • Doherty K.V.
      Flow cytometric analysis of cell division by dilution of CFSE and related dyes.
      ).

      Cell Cycle-Associated Protein Assays

      As cells progress through the cell cycle, phase-specific proteins are generated and can be detected using antibodies. Example phase-specific proteins include topoisomerase II alpha, phosphorylated-histone H3, and proliferating cell nuclear antigen, though there are many others. Topoisomerase II alpha expression begins in late S phase and peaks in the G2 and M phases (
      • Woessner R.D.
      • Mattern M.R.
      • Mirabelli C.K.
      • Johnson R.K.
      • Drake F.H.
      Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.
      ). Histone H3 becomes phosphorylated only during the M phase of the cell cycle (reviewed in
      • Hans F.
      • Dimitrov S.
      Histone H3 phosphorylation and cell division.
      ). Proliferating cell nuclear antigen expression increases during late G1 and peaks during S phase (
      • Kurki P.
      • Lotz M.
      • Ogata K.
      • Tan E.M.
      Proliferating cell nuclear antigen (PCNA)/cyclin in activated human T lymphocytes.
      ). The most commonly assayed cell cycle-associated protein is Ki-67, although it does not actually allow delineation of the different cell cycle phases because it is expressed in G1, S, G2, and M, but absent in the resting phase, G0.
      The major advantage of assaying cell cycle-associated proteins is that multiple techniques can be used: formalin-fixed paraffin-embedded and frozen tissue samples by microscopy, single cell suspensions by flow cytometry, and cell lysates by western blot (though the last will provide only information on a collection of cells rather than an individual cell). This means that these markers can be used to assay proliferation in patient specimens, provided the tissue was fixed immediately after harvesting. Ki-67 is increasingly used to detect proliferating cells in clinical melanoma specimens (
      • Gimotty P.A.
      • Van Belle P.
      • Elder D.E.
      • Murry T.
      • Montone K.T.
      • Xu X.
      • et al.
      Biologic and prognostic significance of dermal Ki67 expression, mitoses, and tumorigenicity in thin invasive cutaneous melanoma.
      ). Figure 3a depicts immunohistochemical staining of Ki-67 together with melanocyte differentiation antigen in a patient’s melanoma specimen (
      • Aris M.
      • Zubieta M.R.
      • Colombo M.
      • Arriaga J.M.
      • Bianchini M.
      • Alperovich M.
      • et al.
      MART-1- and gp100-expressing and -non-expressing melanoma cells are equally proliferative in tumors and clonogenic in vitro.
      ). Figure 3b shows immunofluorescence staining Ki-67 to assess melanocyte proliferation during hair follicle development (
      • Botchkareva N.V.
      • Botchkareva V.A.
      • Gilchrest B.A.
      Fate of melanocytes during development of the hair follicle pigmentary unit.
      ). A second advantage is that researchers can easily stain multiple cell cycle-associated proteins at one time to ascertain a cell’s stage of proliferation. A disadvantage is that no subsequent assays can be performed with cell cycle-associated protein staining because of fixation and permeabilization of the cell’s membranes.
      Figure 3
      Figure 3Example staining and microscopy of Ki-67. (a) Chromogenic staining detected via light microscopy of Ki-67 (brown) and melanocyte differentiation antigen, MD-Ag (red) in melanoma. Arrows depict different staining patterns (Ki-67+MD-Ag, Ki-67+MD-Ag+, Ki-67-MD-Ag+, and Ki-67-MD-Ag). Reprinted with permission from
      • Aris M.
      • Zubieta M.R.
      • Colombo M.
      • Arriaga J.M.
      • Bianchini M.
      • Alperovich M.
      • et al.
      MART-1- and gp100-expressing and -non-expressing melanoma cells are equally proliferative in tumors and clonogenic in vitro.
      . (b) Fluorescent microscopy depicting Ki-67 expression (red) and tyrosinase (green) in a hair follicle during development. Nuclei counterstained in blue (TO-PRO3 staining).
      Reprinted with permission from
      • Botchkareva N.V.
      • Botchkareva V.A.
      • Gilchrest B.A.
      Fate of melanocytes during development of the hair follicle pigmentary unit.
      .

      Cytoplasmic Proliferation Dyes

      A third approach for assessing cell proliferation utilizes cytoplasmic proliferation dyes that are fluorescent chemicals that permeate the cell membrane and covalently bind to cellular cytosolic components (reviewed in
      • Quah B.J.C.
      • Parish C.R.
      New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.
      ). After each cell division, the dye is evenly distributed between the two daughter cells (as a result of cytokinesis), which then exhibit half the fluorescence intensity of their parent cell (Figure 4a) (reviewed in
      • Quah B.J.C.
      • Parish C.R.
      New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.
      ). Cytoplasmic proliferation dyes can be used both in vitro and in vivo. For in vivo use, single cell suspensions are stained with a cytoplasmic dye and then injected into an animal. The cells can then later be harvested from the animal and assayed to determine whether the cells proliferated in vivo. When cytoplasmic dyes are used to assess proliferation, flow cytometry is the assay of choice as it allows visualization of each round of division (Figure 4). Although cytoplasmic dyes can be viewed under microscopy, microscopy does not easily allow quantification of dye and therefore is not typically a reliable means of measuring division.
      Figure 4
      Figure 4Schematic and example data of carboxyfluorescein diacetate succinimidyl ester (CFSE) cytoplasmic proliferation dye assay. (a) Schematic showing decreased CFSE fluorescence intensity exhibited by daughter cells after each successive round of cell division. Each black peak reflects one round of division, and the taller the peak, the more cells underwent division during that round. (b) Flow cytometry histograms demonstrating T-cell proliferation in vivo when intravenously injected into mice 1 or 3 days after that mouse was treated with tolerogenic dendritic cells or not (no DC).
      Reprinted with permission from
      • Divito S.J.
      • Wang Z.
      • Shufesky W.J.
      • Liu Q.
      • Tkacheva O.A.
      • Montecalvo A.
      • et al.
      Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation.
      .
      The classic cytoplasmic dye used in proliferation assays is carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a green fluorescent dye. Because neither CFSE staining nor flow cytometry is an endpoint assay, dyed cells can be sorted and used in further experimentation. One word of caution with CFSE: after enough rounds of division, CFSE fluorescence will no longer be detectable above nonlabeled cells. With optimization, however, the CFSE technique can detect beyond seven rounds of proliferation (
      • Quah B.J.C.
      • Parish C.R.
      New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.
      ). A frequent use of CFSE is to analyze T-cell proliferation. For example,
      • Seneschal J.
      • Jiang X.
      • Kupper T.S.
      Langerin+ dermal DC, but not Langerhans cells, are required for effective CD8-mediated immune responses after skin scarification with vaccinia virus.
      used CFSE staining to demonstrate in vivo that dermal dendritic cells stimulate transgenic T-cell proliferation in response to vaccination, and
      • Divito S.J.
      • Wang Z.
      • Shufesky W.J.
      • Liu Q.
      • Tkacheva O.A.
      • Montecalvo A.
      • et al.
      Endogenous dendritic cells mediate the effects of intravenously injected therapeutic immunosuppressive dendritic cells in transplantation.
      used CFSE to assess T-cell proliferation in response to therapeutic dendritic cells (Figure 4b).
      A drawback of CFSE is that it utilizes the green fluorescent channel, thereby preventing inclusion of additional stains that also require that channel (ie, fluorescein, green fluorescent protein and fluorescein isothiocyanate) (reviewed in
      • Quah B.J.C.
      • Parish C.R.
      New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.
      ). Newer commercially available stains such as CellTrace Violet (ThermoFisher Scientific, Cambridge, MA) are gaining popularity because they fluoresce under a different wavelength of light, thus making available the fluorescein channel for other stains (
      • Quah B.J.C.
      • Parish C.R.
      New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.
      ). CellTrace Violet does, however, require a blue/violet laser that may not be available on all detection equipment.

      Indirect Measures of Cell Proliferation: Cell Counting, Viability, and Metabolic Activity Assays

      Information regarding cell proliferation may be gleaned from cell counting alone or via cell viability stains or assays. The most commonly employed viability stain is trypan blue that is excluded from living cells but absorbed by dead cells (dead cells thus appear blue under light microscopy). Metabolic activity assays use enzymatic activity as a marker for cell viability. The enzyme(s) tested produce a colored or fluorescent product that can be easily measured by a plate reader. Example metabolic activity assays are the tetrazolium assay, resazurin reduction assay, and protease activity assay (reviewed in
      • Riss T.L.
      • Moravec R.A.
      • Niles A.L.
      • Benink H.A.
      • Worzella T.J.
      • Minor L.
      • et al.
      Cell viability assays.
      ). It is important for the reader to understand that none of these methods directly assess cell proliferation. For example, cell counting may suggest that cell proliferation has occurred if there is an increase in the number of cells. However, an alternative explanation could be that cell death decreased. Similarly, an increase in enzymatic activity in a group of cells could indicate increased number of cells due to proliferation, or could reflect increased metabolic activity of nonproliferating cells. Therefore, when interpreting studies using these techniques (like all studies), the reader should ask him or herself, “could anything else account for the observed data” and if so, “do either the presented experimental controls or further experiments address these alternative possibilities”?

      Concluding Remarks

      Table 1 summarizes the most commonly employed cell proliferation assays, including which cell cycle phase the technique assesses and the major equipment needed for that technique. Table 1 also includes recommendations regarding which assay may be most appropriate for a particular experimental application. However, as stated in the introduction, choice of a particular assay is highly individualized based on the experimental objectives, available equipment, and sample type. Clinically, we expect that staining for cell proliferation markers to assess tumor aggressiveness and guide clinical care will likely become standard of care going forward, particularly in the setting of melanoma, where absence/presence of mitotic figures is already part of melanoma clinical staging. This article provides a framework for the investigator and clinician to understand cell proliferation assays, but the reader should know that modifications and optimization of the above assays are ongoing and new techniques are constantly under development.
      Table 1Summary of commonly employed cell proliferation assays including associated cell-cycle phase, equipment, and example recommended applications for each assay
      ApproachAssayCell-cycle phaseMajor equipmentExample recommended application(s)
      Nucleoside-analog incorporationTritiated thymidineSLiquid scintillation counterCell culture, mixed lymphocyte reactions
      Nucleoside-analog incorporation5-Bromo-2′-deoxyuridineSFlow cytometer, light microscope, fluorescence microscopeIn vivo for the study of proliferation over time in animals
      Cell cycle-associated proteinKi-67G1, S, G2, MFlow cytometer, light microscope, fluorescence microscopeHistology
      Cell cycle-associated proteinPhosphorylated-histone H3MFlow cytometer, light microscope, fluorescence microscopeHistology
      Cell cycle-associated proteinProliferating cell nuclear antigenSFlow cytometer, light microscope, fluorescence microscopeHistology
      Cytoplasmic proliferation dyeCarboxyfluorescein diacetate succinimidyl esterCytokinesisFlow cytometerIn vivo proliferation of adoptively transferred stained cells, mixed lymphocyte reactions
      Cytoplasmic proliferation dyeCell trace violetCytokinesisFlow cytometer with a 405-nm laserIn vivo proliferation of adoptively transferred stained cells, mixed lymphocyte reactions

      Conflict of Interest

      The authors state no conflict of interest.

      CME Accreditation

      This activity has been planned and implemented by the Duke University Health System Department of Clinical Education and Professional Development and Society for Investigative Dermatology for the advancement of patient care. The Duke University Health System Department of Clinical Education & Professional Development is accredited by the American Nurses Credentialing Center (ANCC), the Accreditation Council for Pharmacy Education (ACPE), and the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing education for the health care team. Duke University Health System Department of Clinical Education and Professional Development designates this enduring activity for a maximum of 1.0 AMA PRA Category 1 Credits™. Physicians should claim only credit commensurate with the extent of their participation in the activity.
      To participate in the CME activity, follow the link provided. http://continuingeducation.dcri.duke.edu/sid-journal-based-translational-education-dermatology-research-techniques-made-simple-2015

      Multiple Choice Questions

      • 1.
        Which of the following is NOT used for assessing cell proliferation?
        • A.
          Cytoplasmic proliferation dyes
        • B.
          Cell cycle-associated proteins
        • C.
          Nucleoside-analogs incorporated during DNA synthesis
        • D.
          Bioassay-guided fractionation
      • 2.
        Which of the following methods uses radioactive material?
        • A.
          Tritiated thymidine incorporation
        • B.
          Cell counting
        • C.
          5-Bromo-2′-deoxyuridine incorporation
        • D.
          Carboxyfluorescein diacetate succinimidyl ester dye
      • 3.
        In which of the following phases of the cell cycle is Ki-67 expression absent?
        • A.
          G0
        • B.
          G1
        • C.
          S
        • D.
          G2
      • 4.
        Which proliferation assay can be used on patient samples that are formalin-fixed and paraffin embedded?
        • A.
          CFSE labeling
        • B.
          Tritiated thymidine incorporation
        • C.
          Ki-67 staining
        • D.
          BrdU incorporation
      • 5.
        Which assay can be used to measure proliferation in vivo in an animal over time? Which assay provides a snapshot of proliferation at the time of tissue harvest?
        • A.
          Tetrazolium salt assay, Ki-67 staining
        • B.
          Histone H3 staining, CFSE labeling
        • C.
          BrdU incorporation, Ki-67 staining
        • D.
          Ki-67, BrdU incorporation
      This article has been approved for 1 hour of Category 1 CME credit.
      To take the quiz, with or without CME credit, follow the link under the “CME ACCREDITATION” heading.

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