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Conatumumab is a monoclonal antibody specific for death receptor 5 DR5 that activates caspases leading to DNA fragmentation and tumorcell death. Like other Tumor Necrosis FactorRelated ApoptosisInducing Ligand TRAIL receptor therapies, conatumumab is currently being evaluated in clinical trials across a variety of tumor types. However, molecular evidence of ontarget drug activity in tumors is often an elusive goal for clinical investigation. Here we evaluated a translational approach using a relevant biopsy method, fine needle aspirates FNAs, to study the ontarget pharmacodynamics of conatumumab preclinically. As detected by laser scanning cytometry, druginduced caspase3 activation in FNA biopsies of Colo205 xenografts correlated well with activated caspase3 in conventional sectionbased samples. Furthermore, in tumorbearing mice, surrogate assays of serum caspase37 activity and serum drug exposure correlated with in situ caspase3 activation. We found that one advantage of FNA sampling over other sampling techniques was the ability to measure caspase activity on a per cell basis using DNA content information. To adapt the utility of FNAs for measuring pharmacodynamic markers in humans, detection of activated caspase3 was multiplexed with EpCAM to characterize mock and clinical FNAs from colorectal and nonsmall cell lung cancer patients. These data suggest that FNA sampling is a practical method to cytometrically evaluate tumors for pharmacological impact in a clinical setting. © 2010 International Society for Advancement of Cytometry

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals.  Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG1) to human DR5, which induces apoptosis via caspase activation.  In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspasesin vitroin the presence of a cross-linker.  We also show that conatumumab has activity in vivoand inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122), and pancreatic (MiaPaCa2/T2) xenograft models.  Conatumumab also enhances the anti-tumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab.  We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model.  These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

The breast and ovarian cancer susceptibility gene BRCA1encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1−S and persistence of BRCA1 during G2/M. After γ-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.

Mast cells MCs have important functional roles in leukocyte recruitment, pain, and wound healing, and increased tissue resident MC function has been associated with several fibrotic diseases. Consequently, the study of MCs in situcan be a direct approach to studying the pharmacodynamic impact of MCdirected therapeutics in tissues. Here we describe an automated laser scanning cytometry assay that was used to characterize the kinetics of MC accumulation in healing skin wounds and to study the effect of inhibiting CD117 cKit signaling. The number of tryptasepositive MCs approximately doubled 14 days after cutaneous injury in nonhuman primates. Treatment of animals with antiCD117 or imatinib mesylate Gleevec® reduced MC accumulation at the edge of healing wounds in mice and nonhuman primates, respectively. In translating this MC assay to become a biomarker for human studies, no differences in dermal MC numbers were evident between genders, ages or body mass index from 20 healthy donors. These data suggest that skin is a practical and useful tissue for tracking pharmacodynamic effects of MCdirected therapies. © 2008 International Society for Advancement of Cytometry

Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G1 and G2/M) to obtain good quality RNA for gene expression analyses. The experimental design included sorting G1 and G2/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. Sorted living G1 and G2/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle. Cytometry 49:170–175, 2002. © 2002 Wiley-Liss, Inc.

It was observed before that DNAin situin chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stranded (ds) vs single-stranded (ss) nucleic acids, as a marker of the degree of DNA denaturation. However, without prior cell treatment with heat or acid no presence of single-stranded DNA in either mitotic or interphase cells was detected by this assay. In the present experiments we demonstrate that DNAin situin mitotic cells, without any prior treatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal PHA-stimulated lymphocytes with S1 or mung bean nucleases generated extensive DNA breakage in mitotic cells. DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. Under identical conditions of the cells’ exposure to ss-specific nucleases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mitotic loops may lead to formation of the hairpin-loop structures by inverted repeats; such structures are sensitive to ss nucleases. The present method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sensitivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry.

The Programmed Death-1 (PD-1) gene is a member of the immunoglobulin superfamily of genes. Murine PD-1 mRNA expression has been shown to correlate with activation-induced apoptosis in a mouse T-cell hybridoma cell line and in murine thymocytes. Here we report that expression of the human homolog, hPD-1, seems to correlate with activation of T lymphocytes rather than apoptosis. We observed a time-dependent upregulation of hPD-1 mRNA and protein levels in Jurkat cells during phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA)-induced differentiation. Human PD-1 protein was also induced during lectin-stimulated activation of human peripheral blood mononuclear cells. Additionally, TPA stimulation of Jurkat cells induces tyrosine phosphorylation of hPD-1, putatively on its cytoplasmic tail signal transduction motif. These data suggest a role for hPD-1 during activation and differentiation of T-lymphocytes.

A novel methodology for labeling, isolation, and detection of nucleic acids is described. Nucleic acid isolation is based onin vivoorin vitroincorporation of BrU or BrdU to either RNA or DNA, respectively, followed by immunoprecipitation of the labeled nucleic acid utilizing anti-BrdU MoAb, which crossreacts with BrU, attached to solid particles. Filter-bound bromine-labeled DNA or RNA was detected by immunoblotting with anti-BrdU MoAb, by a combined Southern/Western or Northern/Western approach, respectively. This method was applied to isolate and detect rRNA and mRNA from human cells, plasmid DNA from bacterial cells, andin vitrosynthesized DNA. Newly transcribed BrU-labeled mRNA was recovered from the immunoprecipitates and analyzed by RT-PCR to study phorbol ester-mediated regulation of interleukin 1 gene transcription in human leukemic HL-60 or lymphoma U937 cells. The plasmid DNAs were isolated by immunoprecipitation from transformed bacterial cultures that were grown in the presence of BrdU and were detected immunochemically on filters. Likewise, the products of RT-PCR and Klenow polymerase-catalyzed DNA synthesis in which dTTP was replaced with BrdUTP were detected by immunoblotting. Since the method allows one to selectively separate or detect nucleic acids only synthesized during a pulse of the precursor, it can uniquely be used to identify nascent gene transcripts or the transcripts synthesized within specific time windows, e.g., after induction of differentiation, carcinogenesis, or drug treatment, and distinguish such transcripts from preexisting ones. In addition, this approach offers a simple and inexpensive alternative for preparing labeled DNA as well as RNA probes for use in a variety of hybridization protocols. Due to the low toxicity of BrU and BrdU, this approach can be used in analysis of gene transcription or DNA replicationin vivo.

Phosphorylation of the protein encoded by retinoblastoma susceptibility gene (pRb) is the key event of the cell cycle committing the cell to enter S phase and also required for progression through S and G2. We describe a new methodology to monitor pRb phosphorylation in individual cells and correlate it with the cell cycle position. Specifically, pRb phosphorylation in human lymphocytes was assayed immunocytochemically using mAb which recognizes underphosphorylated pRb (pRbP−) conjugated with a fluorochrome of one color combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT) tagged with another color fluorochrome. DNA was stained with still another color fluorochrome and cell fluorescence was measured by multiparameter flow cytometry. Specificity of anti-pRbP−mAb was confirmed by preincubation of the permeabilized cells with phosphatase. Analysis of pRbP−or a ratio of pRbP−/pRbTrevealed that pRb was underphosphorylated in over 98% of the nonstimulated lymphocytes. The proportion of cells with underphosphorylated pRb dropped to 20% between 3 and 8 h after addition of the mitogen phytohemagglutinin (PHA). Phosphorylation of pRb within a cell was rapid and complete since reactivity of individual lymphocytes with anti-pRbP−mAb was lost abruptly rather than step-wise during stimulation. Phosphorylation of pRb coincided with the appearance of cyclin D3, which was induced 3 h and peaked 12 h after addition of PHA. The nonspecific protein kinase inhibitor staurosporine at a concentration known to arrest lymphocytes in G1but not to interfere with the induction of cyclin D3 (20 nM) prevented pRb phosphorylation. The present assay can be applied for screening antitumor drugs targeting CDKs and be useful for monitoring pRb phosphorylation in human tumors, the feature of a possible prognostic value in oncology.

Expression of pRb and its state of phosphorylation were immunocytochemically assayed in individual HL-60 cells during their proliferation and after induction of differentiation, using mAb which detects hypophosphorylated pRb (pRbP-) combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT). Correlated measurements of pRbP-, pRbT, a ratio of pRbP-/pRbT, and cellular DNA content by flow cytometry revealed expression of total pRb and its phosphorylation state vis-à-vis the cell cycle position. Following mitosis (during the exponential phase of cell growth) a mixture of hypo- and hyperphosphorylated pRb was present within the cell for less than 2 h, i.e., early in G1; no hypophosphorylated pRb was detected throughout remainder of the cycle. Cellular pRb content was increasing primarily during G1and the cell entrance to S was correlated with attainment of a distinct threshold level of pRb. No correlation was seen between the content of pRb per cell and its state of phosphorylation during G1. Cell differentiation whether induced by 1,25-dihydroxyvitamin D3, retinoic acid, or phorbol myristate acetate led to cell arrest primarily in G0/1. The G0/1cells in these cultures, compared to G1cells from the untreated cultures, had increased level of both pRbTand pRbP-. However, because the relative increase of pRbP-was disproportionally greater than of pRbT, the pRbP-/pRbTratio of the differentiating cells was markedly elevated. The cells that still were in S and G2/M in the differentiating cultures also showed the presence of hypophosphorylated pRb. Our data suggest that the mechanism of irreversible cell cycle arrest during terminal differentiation involves both the increase in content of pRb and dephosphorylation of pRb already present within the cell. This provides a large pool of hypophosphorylated pRb that can effectively remove all free E2F, thereby precluding activation of the genes whose transcription is needed to pass the G1restriction point. In contrast to terminal differentiation the transient quiescence (G0state) manifests only by dephosphorylation of pRb, without a change in its cellular level.

Phosphorylation of the nucleosome core histone H3 (H3) on Ser-10 is thought to be a prerequisite for chromatin condensation at mitosis. Although during interphase, cell differentiation, or mitogenic activation of quiescent cells, changes in chromatin structure that involve local chromatin condensation/decondensation also occur, little is known about H3 phosphorylation during these transitions. Using the recently developed sensitive marker to monitor H3 phosphorylation, namely, the mAb that recognizes the phosphorylated epitope of H3 (anti-H3-P mAb), the status of H3 phosphorylation was assayed in individual human lymphocytes after their mitogenic stimulation (G0to G1transition) and in human leukemic HL-60 cells induced to differentiate by all-trans-retinoic acid (RA), 1,25-dihydroxyvitamin D3(vit D3), dimethyl sulfoxide (DMSO), or phorbol myristate acetate (PMA). Multiparameter flow cytometry was used to correlate H3 phosphorylation with cell cycle position. The specificity of the anti-H3-P mAb was confirmed by the loss of its binding following cell treatment with alkaline phosphatase. The presence of phosphorylated H3 was detected during interphase in HL-60 cells and in normal lymphocytes at a level severalfold lower than during mitosis. No significant changes in H3 phosphorylation were observed during lymphocyte stimulation. Unexpectedly, the level of H3 phosphorylation was over fourfold higher in monocytes than in lymphocytes or granulocytes from peripheral blood. The punctate pattern of labeling with anti-H3-P mAb in monocyte nuclei suggests that H3 is phosphorylated in small clusters of adjacent nucleosomes. Differentiation of HL-60 cells was accompanied by a rise in H3 phosphorylation, which was higher after induction by RA, vit D3, and PMA (approx. threefold) than after DMSO (∼20%). The data indicate that in addition to being a critical event during chromatin condensation at mitosis, H3 phosphorylation plays a role during chromatin changes accompanying differentiation of HL-60 cells, in particular, along the monocytic lineage. The high level of H3 phosphorylation in monocytes may serve as a marker of these cells and is being explored as a possible diagnostic and prognostic tool in monocytic leukemias.

Phosphorylation of histone H3 (H3) on Ser-10 correlates with chromatin condensation at mitosis. A new monoclonal antibody (anti-H3-P) was developed that recognizes phosphorylated H3 (H3-P). This antibody was used in multiparameter flow cytometric analysis to relate H3 phosphorylation in individual human leukemic cells to the cells' position in the cycle as well as their expression of cyclins A and B1. Mitotic cells, from prophase to telophase, reacted with anti-H3-P; the binding of the antibody to chromatin of interphase cells was several times weaker. Cell growth in the presence of staurosporine, an inhibitor of the kinase(s) that phosphorylate H3, abolished the cells' reactivity with the antibody. The reactivity also was abolished by incubation of permeabilized mitotic cells with alkaline phosphatase. These data indicate that, within permeabilized cells, the antibody is indeed specific for H3-P and does not detect the unphosphorylated epitope. All cells reacting with anti-H3-P, with the exception of prophase and early prometaphase, were cyclin A negative; the expression of cyclin B1 in these cells was threefold higher than in G2 cells. The analysis of phosphorylation of H3 in individual cells when combined with multiparameter analysis of their cycle position and expression of other proteins offers new possibilities to study molecular mechanisms associated with the G2 to M transition and chromatin condensation. It also offers an assay to screen in vivo inhibitors of kinase(s) or phosphatase(s) involved in H3 phosphorylation or dephosphorylation, and it provides a valuable marker to identify mitotic cells by cytometry. Cytometry 32:71–77, 1998. © 1998 Wiley-Liss, Inc.

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology. Cytometry 27:1–20, 1997. © 1997 Wiley-Liss, Inc.

Schuster: Amgen Inc.: Equity Ownership, Honoraria, Speakers Bureau. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Gamelin:Amgen Inc.: Employment, Equity Ownership. Rasmussen:Amgen Inc.: Employment, Equity Ownership. Juan:Amgen Inc.: Employment, Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Chow:Amgen Inc.: Employment, Equity Ownership. Friberg:Amgen Inc.: Employment, Equity Ownership. Vogl:Amgen Inc.: Employment, Equity Ownership.

Background:Aurora kinases form a family of highly conserved serine/threonine protein kinases that regulate key steps in mitosis. Aurora kinases A and B are amplified and/or overexpressed in many malignancies, including various types of leukemia, and are associated with high proliferation rates, poor prognosis, and therapeutic resistance. AMG 900 is an investigational, orally administered, highly potent, selective, small-molecule pan-aurora kinase inhibitor that has shown single-agent activity in heavily pretreated pts with chemotherapy-resistant/refractory solid tumors. This open-label, multicenter, sequential dose escalation study (NCT01380756) assessed AMG 900 in adult pts with AML.

Cheung: Amgen Inc: Employment, Equity Ownership. Juan:Amgen Inc.: Employment, Equity Ownership. Wayne:Amgen Inc.: Employment, Equity Ownership. Hanestad:Amgen Inc.: Employment, Equity Ownership. Keegan:Amgen Inc.: Employment, Equity Ownership. Huard:Amgen Inc.: Employment, Equity Ownership. McElroy:Amgen Inc.: Employment, Equity Ownership. Stanton:Amgen Inc.: Employment, Equity Ownership. Bush:Amgen Inc.: Employment, Equity Ownership. Kendall:Amgen Inc.: Employment, Equity Ownership. Radinsky:Amgen Inc.: Employment, Equity Ownership. Abella:Amgen Inc. : Employment, Equity Ownership. Pieslor:Amgen Inc.: Employment, Equity Ownership. Friberg:Amgen Inc.: Employment, Equity Ownership. Coxon:Amgen Inc.: Employment, Equity Ownership. Gamelin:Amgen Inc: Employment, Equity Ownership. Payton:Amgen Inc.: Employment, Equity Ownership. Off Label Use: AMG 900 is currently in phase 1 clinical development, there is no approved label.

Aurora kinases A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including acute myeloid leukemia (AML) (Ikezoe T et al, 2007). AMG 900, a potent and highly selective small molecule inhibitor of aurora kinases, is currently in Phase 1 clinical testing in adult patients with AML.

Multiphoton‐induced second‐harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three‐dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues. Some technical considerations are also addressed. Curr. Protoc. Cytom. 61:6.33.1‐6.33.11. © 2012 by John Wiley & Sons, Inc.

Cell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G0versus G1cells and to detect cell differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst‐PY method is not applicable to single‐laser instruments. A third protocol describes staining of viable cells to identify/sort hematopoietic stem cells, with an alternative that includes simultaneous immunostaining.

In preparation for cell division, nuclear chromatin undergoes a vital rearrangement required for the organization of chromosomes and their allocation to daughter cells. This process is initiated during G2phase with the most remarkable morphological manifestation being chromatin condensation. This unit provides protocols for identification and quantification of mitotic cells based on immunocytochemical detection of histone H3 phosphorylated on Ser 10 (H3‐P), the critical event occurring during the G2to M transition (essential for chromatin condensation), using anti‐H3‐P, a commercially available antibody to which apoptotic cells are not reactive, concurrently with differential staining of cellular DNA. Additionally an adaptation of this method used to stain cells mounted on microscope slides for analysis by multiparameter laser scanning cytometry is also presented.

This unit provides a detailed method for evaluating in situ DNA denaturation by flow cytometry. The principal technique is based upon the metachromatic properties of acridine orange. This technique has a number of advantages over traditional biochemical methods, but requires very precise methodology; the result is excellent clarity and differentiation between single‐stranded and double‐stranded DNA. This unit provides full details on the correct use of acridine orange together with an excellent discussion of the pitfalls and problems.

This unit describes a method for identifying apoptotic cells based on in situ detection of DNA strand breaks. Correlated analysis of DNA content and DNA strand breaks allows one not only to identify apoptotic cells but also to pinpoint their location in the cell cycle. The basic protocol uses 5-bromodeoxyuridine triphosphate to label the 3-OH termini of the breaks, but the procedure is easily adapted to a variety of other conjugates. Given the multiplicity of commercially available reagents it is possible to label DNA strand breaks with a dye of any fluorescence color and excitation wavelength.

This unit presents the most common methods of DNA content analysis. The techniques differ primarily in the mode of cell permeabilization (detergents versus prefixation with alcohol) and composition of the stain solution. Protocols are given for analysis of fixed cells, tissue samples, live cells (for subsequent sorting and culture), archival paraffin-embedded tissue, and apoptotic cells.

Cell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G0 versus G1 cells and to detect cell differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst-PY method is not applicable to single-laser instruments.

This unit discusses the general applicability of nucleic-acid analytical methods in both basic and clinical research. After immunophenotyping, analysis of nucleic acids is perhaps the most common application of flow cytometry. The methodologies are of particular interest for researchers in basic and clinical oncology, as they provide information on cell attributes associated with tumor malignancy, and are widely used in various areas of cell and molecular biology.

During the past two decades, the techniques of analysis of cell cycle kinetics based upon the flow cytometric detection of BrdU incorporation have replaced the cumbersome autoradiographic methodology utilizing tritiated thymidine. This unit distills the knowledge gained over this time into concise and easy to follow protocols for DNA content analysis following thermal denaturation or acid denaturation, as well as a modified method that combines thermal denaturation with washless immunocytochemical detection of BrdU.

Cyclins are key components of the cell cycle machinery. This unit describes techniques associated with measurement of these transiently expressed molecules and focusses on the combination of cell cycle analysis and expression of proliferation-associated proteins via immunocytochemically attached antibodies.

This unit describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. These staining procedures can be used with fixed cells or detergent‐permeabilized cells. Alternatively, live cells can be stained with the membrane‐permeable stain, Hoechst 33242, and sorted on the basis of DNA content for further culture and characterization. When DNA content measurements are coupled with immunostaining for cyclin expression, it is possible to reliably detect even more stages in progression through the cell cycle.