BD Biosciences Research Grants

Winter 2015 Research Grant Recipients Talk About Their Research


Taylor Brost
Graduate Student
Washington University in St. Louis School of Medicine

Abstract Title:
Interrogation of Clonal Evolution in Myeloproliferative Neoplasms Utilizing Patient-Specific Induced Pluripotent Stem Cells


BD: What is your educational background?

Taylor Brost: I earned my BS degree from Minnesota State University in human biology with a minor in chemistry. During my summers as an undergraduate I worked in Dr. J. Carter Ralphe’s laboratory at the University of Wisconsin, studying hypertrophic cardiomyopathy. I am currently a PhD student at Washington University in St. Louis School of Medicine in the division of biology and biomedical sciences, in the molecular cell biology program.

BD: How and when did you become interested in science?

Taylor Brost: I have been interested in science for as long as I can remember. The human body has always fascinated me, especially at the cellular level. My interest was amplified in my high school biotechnology class where I started to learn scientific methods and how experiments can lead to new knowledge. I had an amazing teacher for the course who commended and encouraged my continuous questions.

BD: How did you become involved in your broad field of study?

Taylor Brost: I was introduced to working with stem cells in Dr. Ralphe’s lab as an undergraduate. I quickly realized the powerful model system that stem cells provide. While I was considering laboratories for my PhD thesis work, I looked for a lab that provided the opportunity to learn more about stem cells and their utility as a disease model system. My hematology course in college sparked my interest in hematologic malignancies, and I was privileged enough to find a lab that allows the opportunity to utilize induced pluripotent stem cells (iPSCs) to study clonal evolution in blood cancers.

BD: Describe the project for which you received the BD grant.

Taylor Brost: Myeloproliferative neoplasms (MPNs) are chronic myeloid malignancies clonally derived from hematopoietic stem or progenitor cells (HSPCs). They are characterized by an overproduction of one or more myeloid lineages as a consequence of unrestrained cellular proliferation. The JAK2 V617F mutation has been identified in all three classic MPNs (polycythemia vera, essential thrombocythemia, primary myelofibrosis), indicating a shared hallmark of JAK-STAT signaling dysregulation in MPNs. However, the precise role for JAK2 V617F in dictating specific MPN phenotypes remains unresolved. While targeted inhibitors of JAK2 provide symptomatic benefit for MPN patients, they are not effective in eradicating the malignant clone, and they do not prevent transformation to secondary acute myeloid leukemia. Therefore, the specific role of the JAK2 V617F mutation in MPN pathogenesis remains incompletely understood.

This work seeks to uncover MPN disease mechanisms by developing human models to investigate MPN pathogenesis. An obstacle to understanding disease mechanisms in MPNs is the inability to maintain primary cultures of patient-derived MPN specimens. Therefore, we propose to utilize patient-derived iPSCs differentiated along specific hematopoietic lineages to interrogate the role of JAK2 V617F and other driver mutations, in hopes of elucidating how clonal evolution influences MPN disease initiation, development and progression. The initial focus of this project is to determine whether JAK2 V617F is sufficient and/or required to induce the MPN phenotype observed in JAK2 V617F-mutant patients. We hypothesize that driver mutations in addition to JAK2 V617F contribute to the development of specific disease phenotypes.

CD34+ cells from MPN patients and normal donors isolated by fluorescence activated cell sorting have been reprogrammed using the non-integrative Sendai virus method, with the goal of isolating genetically distinct clones harboring specific mutation combinations from individual patients. iPSCs have successfully been generated from two MPN patients, including confirmed JAK2 V617F wild-type, heterozygous and homozygous clones isolated from a single patient. These iPSC clones will undergo directed differentiation toward the definitive hematopoiesis program, enabling the analysis of various lineages shown to be dysregulated in MPNs such as the megakaryocyte and erythroid lineages. Colony-forming assays will also be utilized to study self-renewal and differentiation capabilities of the hematopoietic progenitors generated through directed differentiation.

Mass cytometry will be utilized to assess and uncover signaling profile abnormalities of the patient-specific iPSC clones. These studies will enable the interrogation of altered hematopoietic development and signaling of genetically distinct iPSC clones. These approaches will also be coupled with CRISPR-mediated gene editing to further delineate the contribution of JAK2 V617F and other driver mutations to MPN disease initiation and development.

BD: What are the long- and short-term scientific goals of this project?

Taylor Brost: The short-term goals of this project are to differentiate the patient-specific iPSCs derived from MPN patients that have various mutation profiles to establish differences in phenotypes. The JAK2 V617F homozygous, heterozygous and wild-type clones will be differentiated down the definitive hematopoietic program to determine the functional and signaling abnormalities associated with the mutation. Long term we hope to use the site-directed gene editing technique, CRISPR, to revert and/or introduce driver mutations that contribute to MPN pathogenesis to elucidate how the mutations in isolation and in combination contribute to the MPN disease phenotype.

BD: What are the implications of your project for human health?

Taylor Brost: MPNs cause substantial morbidity and mortality. The median survival in PMF patients is estimated at 6 years, and a subset of patients live only months after diagnosis. PV and ET patients are at risk of transformation to myelofibrosis, and all three malignancies have the potential to transform into secondary acute myeloid leukemia. Ruxolitinib has been developed as a treatment for myelofibrosis, but it does not eradicate the malignant clone, reduce bone marrow fibrosis or increase overall survival of the patients. More effective treatments are necessary. The patient-specific iPSC model system allows for the interrogation of the role of the JAK2 V617F mutation as well as other mutations via hematopoietic differentiation, self-renewal assays and mass cytometry. Gaining an understanding of the signaling abnormalities associated with genetically distinct iPSC clones will potentially uncover novel therapeutic targets.

BD: Which BD reagents do you plan to use, and for what purposes?

Taylor Brost: BD plastic ware will be an important resource, including conicals, pipets, cell strainers, and sort collection tubes. Plastic ware is used on a daily basis and is necessary for the majority of my experiments. BD antibodies will be used to sort HSPCs from normal bone marrow donors as well as from patient peripheral blood or bone marrow, to be reprogrammed into iPSCs. BD antibodies are also conjugated to heavy metal tags for signaling analysis via mass cytometry.


Francesca Chiarini, PhD
Research Scientist
Institute of Molecular Genetics, National Research Council (Italy)

Abstract Title:
Improving Nelarabine Efficacy in Refractory/Relapsed T-ALL


BD: What is your educational background?

Francesca Chiarini: I received my undergraduate degree in biological sciences from the University of Bologna, defending a thesis on gene therapy. My research focused on the efficacy of prophylactic and therapeutic cancer cell vaccines in mice transgenic for the rat Her2/neu oncogene. From January 2006 I began work in the Dept. of Anatomical Sciences, University of Bologna, where I studied innovative targeted anti-tumor therapies in models of human acute leukemias.

Between 2007 and 2009 I was a PhD student in human morphological and molecular sciences. During this time I was involved in investigating signal transduction pathways as innovative targets for the therapy of acute leukemias. As a post-doctoral fellow from January 2010, I was responsible for performing most of the translational studies which were an integral part of a phase 2 national clinical trial (GIMEMA, AML1107, NCT00775593), in which elderly patients with acute myelogenous leukemia (AML) were being treated with a combination of temsirolimus, an allosteric mTOR inhibitor, and clofarabine, a chemotherapeutic drug at low dosage.

BD: How and when did you become interested in science?

Francesca Chiarini: While still in high school I became attracted to biology and also astronomy. My teacher knew how to create interest, and her lessons were really inspiring. From that point forward I knew I wanted to be a biologist.

BD: How did you become involved in your broad field of study?

Francesca Chiarini: My interests in oncology began while I was a master's degree student, through oncology and hematology courses. I remain happy with this choice. During my doctoral work I was involved in the study of novel therapeutic strategies in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Today my daughter Alice is two years old, and when I look at her, I sense the importance of our work. I hope to contribute at least a little toward these young patients.

BD: Describe the project for which you received the BD grant.

Francesca Chiarini: The introduction of novel chemotherapy protocols has improved the outcome of T-ALL patients, but refractory and/or relapsing disease remains a concern. A major contribution was provided by the introduction of nelarabine, approved for salvage treatment of refractory/relapsed T-ALL patients. However, nelarabine induces dose-dependent neurotoxicity. Moreover, patients could develop resistance to nucleoside analogs. To improve nelarabine efficacy, I have analyzed the therapeutic potential of the combination of nelarabine with the phosphoinositide 3-kinase (PI3K) inhibitor ZSTK-474 on both T-ALL cell lines and pediatric patient samples at relapse, displaying constitutive activation of PI3K signaling. Indeed, upregulated PI3K is a common feature of T-ALL, being detectable in 70–85% of the patients, in whom it portends a poorer prognosis by influencing leukemic cell proliferation/survival/drug resistance. Most of the effects of PI3K are mediated by its downstream target Akt.

The combination inhibited the growth of T-ALL cells, and was synergistic in decreasing cell survival and inducing apoptosis. Moreover the combination caused Akt dephosphorylation (inactivation), and a downregulation of pro-survival Bcl2, while nelarabine alone induced an increase of p-Akt (activation) and Bcl2 signaling in resistant to nelarabine T-ALL cell lines and relapsed patient samples. These observations suggest the possibility of combining nelarabine together with specific inhibitors of the PI3K signaling pathway, to improve the efficacy of T-ALL treatment of relapsed/refractory patients.

Nelarabine is rapidly metabolized in the plasma, by an adenosine deaminase, into the active metabolite Ara-G, the latter having a much longer plasma half life and reaching higher plasma concentrations. Ara-G is taken up by leukemia cells via the nitrobenzylthioinosine-sensitive nucleoside membrane transporter equilibrative nucleoside transporters 1 and 2 (ENT1/2). Ara-G is then phosphorylated to Ara-G monophosphate by the deoxycytidine and deoxyguanosine kinases. It is then phosphorylated to its triphosphate form Ara-GTP, which in turn competes with dGTP for DNA polymerase and is subsequently incorporated into DNA, resulting in termination of DNA synthesis.

It has been reported that the levels of expression of ENT 1/2 nucleoside transporters were related to in vitro nelarabine sensitivity of T-ALL cell lines and primary samples. However, no genetic evidence of their involvement was presented. Moreover, the expression of ENT1/2 transporters could be dependent on interactions between leukemic cells and tumor microenvironment. We will study nelarabine metabolism in sensitive and resistant T-ALL cells to understand if it is another mechanism implicated in drug resistance.

BD: What are the long- and short-term scientific goals of this project?

Francesca Chiarini: We expect this project will shed light on the roles played by the PI3K/ mTOR signal transduction pathway in influencing nelarabine sensitivity in T-ALL cells. Additionally, we may discover novel patient-tailored therapeutic approaches through ex vivo testing of primary T-ALL cells, which should delineate individual signaling activation profiles on one side and metabolic phenotypes on the other side. The outcome of this work could have important therapeutic implications, since the signal transduction modulators which will be assessed are already being tested in clinical trials for leukemia patients. Therefore, the findings emerging from our efforts could be rapidly translated into clinical testing in patients with refractory/relapsed T-ALL.

BD: What are the implications of your project for human health?

Francesca Chiarini: Children and adults with relapsed/refractory T-ALL represent a subgroup of patients at higher risk, showing worst outcome despite the modern therapeutic strategies. Several research groups are trying to increase patient survival and reduce toxicities by identifying subgroups of T-ALL responsive to target therapy, to tailor treatment basing on specific biological features of disease. Besides the necessity of introduction of new, improved therapeutic strategies, up to now almost none of the molecular target therapies has been added to standard chemotherapy because of lack of biological studies supporting their safety and efficacy in T-ALL. Thus data obtained from this project could be useful to think of and design new clinical trials for pediatric and adult T-ALL, guided by genetic-specific signatures of patients and biological studies of signaling pathways involved in the sensitivity of therapies.

BD: Which BD reagents do you plan to use, and for what purposes?

Francesca Chiarini: We will make extensive use of flow cytometry, especially in primary samples. I will employ a panel of fluorescently conjugated monoclonal antibodies from BD. We will use primary conjugated antibodies to human cell surface markers and signaling molecules (phosphoproteins) plus BD's antibodies for apoptosis and cell cycle analyses (Caspases, PARP, CFSE, PI).


Gerolama Condorelli, MD, PhD
Associate Professor
Federico II University of Naples, Italy

Abstract Title:
Cancer-Associated Fibroblasts (CAFs) Release Exosomal microRNAs that Dictate an Aggressive Phenotype in Breast Cancer Cells


BD: What is your educational background?

Gerolama Condorelli: I received my MD degree summa cum laude from the University of Naples Medical School in 1988. I was a fellow at the CNR Experimental Endocrinology and Oncology Institute (IEOS) from 1989 to 1991, and a post-doctoral fellow at the Joslin Diabetes Center, Harvard Medical School. In 1995 I received my PhD in cellular and molecular biology and pathology at the Federico II University of Naples, Italy, and became certified for endocrinology and metabolic diseases in 1997. From 1995 to 2002 I was a research associate at the CN.R at IEOS in Naples, Italy. I was then promoted to associate professor of general pathology, in the Department of Molecular Medicine and Medical Biotechnology at Federico II University.

BD: How and when did you become interested in science?

Gerolama Condorelli: Many members of my family are doctors and conduct research on human diseases. Therefore I grew up with “science and milk.” With all the difficulties of this kind of work, I am still enthusiastic and try to transmit this passion to my students.

BD: How did you become involved in your broad field of study?

Gerolama Condorelli: I became interested in stem cells and in particular cancer stem cells (CSCs) during the last five or six years. My interest was influenced by the recent discovery that CSCs comprise a small part of the heterogeneous tumor cell population possessing self-renewal and multi-lineage differentiation potential as well as a great ability to sustain tumorigenesis. CSCs beget new CSCs and simultaneously produce differentiated mature cells responsible for a tumor’s cellular heterogeneity. CSCs are now considered the driving force behind cancer. In fact, they are the only cells able to regenerate a new tumor when xenografted into mice, even when only very few cells are injected. Furthermore, CSCs are resistant to conventional chemotherapy and are considered responsible for tumor recurrence.

BD: Describe the project for which you received the BD grant.

Gerolama Condorelli: Breast cancer is the most common cancer in women. Tumor epithelial cells coexist in carcinomas with different stromal cell types that together create the microenvironment of cancer cells. Cancer-associated fibroblasts (CAFs), the major components of tumor stroma, are active fibroblasts that, similarly to myofibroblasts, are highly heterogeneous, acquire contractile features and express α-smooth-muscle actin. Active fibroblasts play similar roles in wound healing and in cancer, which may be considered as a wound that does not heal. CAFs release high levels of growth factors, cytokines, chemokines and metalloproteases that may affect either other stroma cells or cancer cells. Accumulated evidence indicates that they play an important role in initiation, angiogenesis, invasion and metastasis of breast cancer. Thus, CAFs represent an attractive target for cancer therapy. Exosomes are small (40–100 nm) vesicles that have emerged as important mediators of intercellular communication in cancer. Exosomes control local and systemic cell communication through the horizontal transfer of information, such as microRNAs (miRs), mRNAs and proteins. Over the last decade, a number of studies have revealed that exosomes influence major tumor-related pathways, such as invasion, migration, epithelial-to-mesenchymal transition (EMT), metastasis and therapy resistance. Therefore, we will investigate whether the release of CAF exosomes and their specific miR cargo could dictate an aggressive phenotype in breast cancer.

BD: What are the long- and short-term scientific goals of this project?

Gerolama Condorelli: Short term we hope to analyze the role of CAF exosomes and their miRs in breast cancer progression and in the “stemness” phenotype. We will assess this through in vitro assays on different breast cancer cell lines (estrogen receptor positive and triple negative cells) and on different breast cancer primary cells. In particular, we will investigate the role of microRNAs released from CAF exosomes in: stemness maintenance (mammosphere formation assay, measure of mammosphere diameters, PKH26 assay, re-plating assay), migration (scratch assay, transwell migration assay), proliferation (colony assay, MTT assay) and chemotherapy resistance (MTT assay, caspase assay, fluorescence activated cell sorting analysis). Long term we expect to verify the role of these miRs through in vivo experiments by studying in an orthotropic model of breast cancer the metastatic and angiogenic processes promoted by these miRs.

BD: What are the implications of your project for human health?

Gerolama Condorelli: The importance of this proposed project relates to establishing the role of CAF exosomes-released miRs on the maintenance and expansion of cancer stem cells. The crosstalk between cancer cells and their surrounding microenvironment is essential for tumor progression and the metastatic process. Tumor stroma and tumor cells communicate not only through classical paracrine signaling mechanisms (cytokines, chemokines, growth factors), but also through exosomes. In the context of cancer, this process entails the transfer of cancer-promoting cellular contents between cancer cells and stromal cells within the tumor microenvironment or into the circulation to act at distant sites, thereby enabling cancer progression. Therefore, understanding the molecular mechanisms underlying tumor-microenvironment crosstalk may help the formulation of a specific target therapy and biomarker selection for human cancer.

BD: Which BD reagents do you plan to use, and for what purposes?

Gerolama Condorelli: We will be using proliferation assay reagents, apoptosis reagents, antibodies, flow cytometry kits, magnetic cell separation, ELISA kits and cell culture medium. We plan to use these kits to analyze the composition of fibroblast conditioned media after breast cancer exosome treatment, for example the BD OptEIA™ Human IL-8 ELISA Set, BD OptEIA™ Human IL-6 ELISA Set, BD OptEIA™ Mouse TNF (Mono/Mono) ELISA Set and others.


Le Min, MD, PhD
Assistant Professor
Brigham and Women’s Hospital, Harvard Medical School

Abstract Title:
Evaluating Differences in Lymphocyte Subsets and Gene Expression Profile in Snti-PD1 Responders and Non-Responders


BD: What is your educational background?

Le Min: I received my MD degree from Chongqing Medical University, Chongqing, China, in 1985. This was followed by a master’s degree in pharmacology in 1998, also at Chongqing, and a PhD in pharmacology from the National University of Singapore. From 1999 to 2005 I was a postdoctoral associate at the University of Iowa, followed by an internship in internal medicine, from 2004 to 2005, at the Mount Sinai School of Medicine. I spent three years as a research/clinical fellow in endocrinology under the tutelage of Dr. Ursula Kaiser at Brigham and Women’s Hospital, Boston, specializing in endocrinology.

BD: How and when did you become interested in science?

Le Min: In fact my parents were physicians who practiced both Western and Chinese medicine in China. My parents constantly faced the dilemma of whether to treat their patients with Western or Chinese medicine. I was curious to know the difference between the two types of medical practices. In general, Chinese medicine is based on the old theory that our bodies maintain balance under the regulation of Yin and Yan. Imbalances of Yin and Yan resulted in disease. Further, a more complicated regulation system by five elements (gold, wood, water, fire and earth) help to fine-tune the balance. Herbs and acupuncture commonly used in Chinese medicine help to rebalance the regulation system. On the other hand, Western medicine is based on the evidence from the anatomy, biochemistry, physiology, and pharmacology through clinical studies. I think Western medicine provides a more solid base for overcoming disease, while Chinese medicine may provide some guidance—in theory—on how to apply Western medicine.

BD: How did you become involved in your broad field of study?

Le Min: Immune checkpoint inhibition therapy has emerged as a promising method to overcome advanced malignancies. As a result, it was named “Breakthrough of the Year” by the journal Science in 2013. Because checkpoint inhibition is a form of immunotherapy, autoimmunity increases as well. As an endocrinologist, I have opportunities to help manage patients who receive immune checkpoint inhibition therapy and subsequently develop endocrine disorders. While managing their endocrine disorders, I realized that only about 30% of the patients responded to the immunotherapy. To me, the question was why the remaining patients did not respond. Understanding the mechanisms underlying the responders and non-responders may help to switch a non-responder to a responder.

BD: Describe the project for which you received the BD grant.

Le Min: In human studies, immune checkpoint inhibition induces changes in immunologic profiles. There have been no studies to compare such changes between immune checkpoint blockade responders and non-responders. To characterize the unique immune profiles in responders and non-responders, patients with advanced malignancies who are scheduled to receive anti-PD1, a monoclonal antibody that blocks the immune checkpoint PD1, will be enrolled in our study to evaluate the immune profile change in the responders and non-responders. Blood samples will be collected prior to treatment and at 1, 8, and 24 weeks after the first treatment. The rationale for this proposed timing of blood collection is to look at the immediate response, at the time of peak adverse events (8 weeks) and after tumor response can be fully assessed to identify responders and non-responders (24 weeks). The criteria for responders and non-responders will follow the Response Evaluation Criteria in Solid Tumors (RECIST) protocol. Plus we will measure all patients’ immune profiles.

BD: What are the long- and short-term scientific goals of this project?

Le Min: The long-term goal is to identify the factors that determine who will respond to the therapy. The short-term goal is to evaluate immune profile changes before and after treatment. The findings may help to identify who will respond to the immune checkpoint blockade prior to initiating the treatment. In addition, it may help to convert a non-responder to a responder after we understand the difference between the two immune profiles.

BD: What are the implications of your project for human health?

Le Min: The results of the baseline profiles and the changes in immune profile after the first treatment may reveal significant differences between responders and non-responders, which may provide guidance for identifying responders and non-responders before or shortly after initiation of the immunotherapy. As a secondary goal, by correlating the findings with the occurrence of autoimmune adverse events, the results of this study may also have applications to advance our understanding of immune-related gene profiles in autoimmunity.

BD: Which BD reagents do you plan to use, and for what purposes?

Le Min: We will use the BD™ Human Regulatory T lymphocyte separation set and BD IMag™ Human B Lymphocyte, T lymphocyte, CD8, CD4, monocyte, and NK cell enrichment sets.


Erik Müllers, PhD
Senior Research Scientist
Karolinska Institutet, Stockholm

Abstract Title:
FRET-Activated Cell Sorting (FRACS) as a Novel Setup to Study DNA Damage Checkpoint Recovery


BD: What is your educational background?

Erik Müllers: I earned my undergraduate degree in molecular bioengineering from the Technische Universität Dresden, in Germany. I then studied for my PhD in cell biology and virology in the lab of Prof. Dirk Lindemann, also at the Technische Universität Dresden. I spent time as a postdoc studying cell cycle and DNA damage in the lab of Prof. Arne Lindqvist at the Karolinska Institutet, Stockholm, Sweden.

BD: How and when did you become interested in science?

Erik Müllers: As kid I owned many books of a series called Was ist was (What is what). These are books for children about different science topics, for example, the oceans, astronomy, robots, ancient Egypt, the human body etc. I loved these books. I think I must have acquired more than fifty of them. They sparked my interest in science. And thus, when other children wanted to become firemen or astronauts I wanted to become a scientist.

BD: How did you become involved in your broad field of study?

Erik Müllers: For my PhD I studied a special kind of retrovirus. These viruses have basically only four components, and at the time I naively thought, if we can’t figure out a system of four components, then we don’t even need to try more complex systems. To study the viral life cycle, I developed live-cell fluorescence assays, both for microscopy and flow cytometry. During my postdoc I applied these techniques to the exciting field of cancer cell biology, more precisely the cell cycle and the DNA damage response.

BD: Describe the project for which you received the BD grant.

Erik Müllers: Several cancer-causing mechanisms such as oncogene activation, replication stress, or acquiring of mutations initially lead to activation of cellular checkpoint pathways, causing a robust cell cycle arrest. At some stage however, developing tumor cells somehow overcome this arrest and recover from the checkpoint. Yet, mainly due to technical challenges, our understanding of the underlying molecular mechanisms of checkpoint recovery is very poor. First, due to the inherent asynchrony of checkpoint recovery, it has not been studied except by forced shutdown of a checkpoint. This is a major limitation in the field. Moreover, since not all cells recover from an arrest, spontaneous recovery is an extremely rare event, so that recovering cells make up only a very small fraction of the cell population. Finally, there are currently no known marker proteins to identify cells undergoing recovery.

We devised a method using a biosensor based on Förster-resonance energy transfer (FRET) that allows us to monitor the activity of a protein essential for checkpoint recovery. This tool allows us to identify individual, recovering cells. Using our BD FACSAria™ III system, we recently succeeded in sorting populations of recovering cells according to their individual FRET-ratio—a method we like to call FRET-activated cell sorting (FRACS). Thus, we are now able to precisely identify and collect spontaneously recovering cells.

BD: What are the long- and short-term scientific goals of this project?

Erik Müllers: The potential to collect larger amounts of rare, spontaneously recovering cells opens up new avenues for our research and for the first time allows studying how unsynchronized populations of cells recover from a DNA-damage–mediated arrest. Initially, we will investigate the molecular differences between unperturbed mitotic entry and checkpoint recovery to better understand a key step in early tumor development. Since we can obtain very accurate and robust FRET measurements in fluorescence activated cell sorting, we plan to use this setup as a basis for drug and/or siRNA screens of modulators of checkpoint recovery competence. Additionally, the application of FRET-based biosensors in cell sorting has a tremendous potential in other areas of cell biology research and in large-scale screening approaches.

BD: What are the implications of your project for human health?

Erik Müllers: Despite the large redundancies within the unperturbed mitotic entry network, several proteins become essential for spontaneous recovery from a DNA-damage–induced checkpoint arrest. If we can identify these essential components, we will be able to shed new light on a fundamental biological process that is also a key event in early tumor development. Understanding the mechanisms essential for cancer development might also lead to new target hypotheses for cancer drug discovery.

BD: Which BD reagents do you plan to use, and for what purposes?

Erik Müllers: Our FRACS-based approach for the first time allows us to identify and collect large amounts of cells undergoing spontaneous recovery from a DNA damage arrest. To precisely characterize these cells we utilize protein biochemistry as well as flow cytometry, since it provides us with the possibility to assess cell cycle kinetics and to analyze multiple targets at a time while preserving single-cell resolution. To this end, we rely on a wide range of BD antibodies that allow us to assess key proteins of the mitotic entry network (for example, Cyclin A2, Cyclin B1, Cdk1, Cdk2, Plk1), the DNA-damage network (for example, p53, Kap1, p38) and, most importantly, antibodies for specific posttranslational modifications (for example, H2AX pS139, p53 pS37, Rb pS780, PLK1 pT210, or Cdk1 pY15).