Download or read book Study of Early Signaling Events in T Cell Activation Enabled Through a Modular and Multi time Point Microfluidic Device written by Catherine Aurelie Rivet and published by . This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Binding of the antigen receptor on T cells initiates a rapid series of signaling events leading to an immune response. To fully understand T cell mediated immunity, underlying regulatory properties of the receptor network must be understood. Monitoring dynamic protein signaling events allows for network analysis. Unfortunately, dynamic data acquisition is often extremely time-consuming and expensive with conventional methods; the number of proteins monitored at the same time on the same sample is limited. Furthermore, with conventional, multi-well plate assays it is difficult to achieve adequate resolution at sub-minute timescales. Microfluidics is a capable alternative, providing uniformity in sample handling to reduce error between experiments and precision in timing, an important factor in monitoring phosphorylation events that occur within minutes of stimulation. We used a two-module microfluidic platform for simultaneous multi-time point stimulation and lysis of T cells to investigate early signaling events with a resolution down to 20 seconds using only small amounts of cells and reagents. The device did not elicit adverse cellular stress in Jurkat cells. The activation of 6 important proteins in the signaling cascade upon stimulation with a soluble form of Î"--CD3 in the device was quantified and compared under a variety of conditions. First, in comparison to manual pipetting, the microdevice exhibits significantly less error between experiments. Secondly, a comparison between Jurkat cells and primary T cells shows similar dynamic trends across the 6 proteins. Finally, we have used the device to compare properties of long-term vs, short-term cultured primary T cells. As expected, older cells present a much weakened response to antigenic cues, as measured with TCR response markers. This modular microdevice provides a flexible format for investigating cell signaling properties through the use of soluble cue stimuli.
Download or read book Early Signaling Events in T Cell Activation written by Nancy Chiu and published by . This book was released on 1997 with total page 250 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Interrogating Immune Signaling at Single cell Resolution Using Microfluidic Systems written by Meiye Wu and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Mammalian cell populations are heterogeneous and plastic in their response to stimuli and communication with each other. Multi-parameter quantitative experimental and computational methods that preserve single-cell resolution in these complex cell populations are necessary for elucidating the highly dynamic mechanisms that underlie cell signaling. This thesis explores various aspects of immune cell signaling using integrated microfluidic lab-on-a-chip technologies, capable of generating such multi-parameter single-cell measurements. A brief survey of existing single-cell resolution techniques and their applications in cell signaling studies, including microfluidic devices that evolved into those used in this thesis is discussed. Three microfluidic methods are developed for studying innate and adaptive immune cell signaling. The first method enables the spatiotemporal profiling of the toll-like receptor 4 (TLR4) pathway using a microfluidic device that seamlessly integrates cell culture, sample preparation, imaging and flow cytometry capabilities. Systems-level profiling of signaling proteins in the TLR4 pathway is performed in a single experiment on the integrated microfluidic platform, to generate data used for subsequent mathematical modeling. Next, a new single-cell resolution microfluidic method that combines metabolic labeling of bioorthogonal probes and proximity ligation assay to measure dynamically glycosylated proteins, a post-translational modification with widespread significance in cell signaling modulation, is demonstrated in T cells. The third method extends the utility of the microfluidic platform to measure microRNAs concurrently with proteins in intact T cells, to further expand the repertoire of signaling molecules that can be multiplexed and quantified at single-cell resolution. The lab-on-a-chip technologies developed in this thesis present potential uses for several important applications including basic research, drug and biomarker discovery, biodefense, and for development of cost-effective point-of-care clinical diagnostics.
Download or read book Design and Optimization of Efficient Microfluidic Platforms for Particle Manipulation and Cell Stimulation in Systems Biology written by Alison Marie Paul and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: The overall goal of this research was to develop an efficient microfluidic system to study signal transduction in stimulation dynamics. This research applied reactive transport fundamentals in concert with biological systems knowledge to completely understand diffusion of soluble signals, fluid and particle flow properties, and dynamics of cellular responses. First, a device capable of parallel multi-time-point cell stimulation and lysis on-chip was developed in collaboration. Second, to understand flow of cells through complex 3-D flow schemes, a Single-field Three-dimensional Epifluorescence Particle (STEP) imaging technique was developed. Using the STEP imaging technique, we were able to determine particle distributions and track individual particles in complex flow geometries. Third, during the design of the stimulation device it was observed that the cells do not distribute across the channel in the same way as the fluids. Based on the observation that geometry and particle size were most influential factors on particle distribution, it was hypothesized that our earlier observation and all observed phenomena in our experimental range were due to the volume exclusion of particles of finite size near the wall of the complex flow geometry. Overall, this work contributed to the realization of microfluidic platforms as powerful tools for probing areas of biology and medicine that are difficult with existing technology. The high-throughput format enabled simple and fast generation of large sets of quantitative data, with consistent sample handling. We demonstrated the necessary first steps to designing efficient unit operations on cells in microfluidic devices. The model can be used for informed design of unit operations in many applications in the future.
Download or read book T Cell Receptor Signaling written by Chaohong Liu and published by . This book was released on 2020 with total page 296 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume provides current and new advanced methods and protocols to study T cells. Chapters guide readers through T cell diversity using mass cytometry, analyzing T cells from single cell level, CRISPR/Cas9 techniques to study the T cell activation, techniques to study subsets of Tcell's, procedures to study artificial antigen presentosomes for T cell activation, techniques to study the T cell development, two-photon microscopy, and MAIT cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, T-Cell Receptor Signaling: Methods and Protocols aims to provide a wide range of approaches and be an invaluable resource for present and future generations of T cell researchers.
Download or read book Regulatory Mechanisms of Early Intracellular Signaling in T Lymphocytes written by Enrique Aguado and published by Frontiers Media SA. This book was released on 2021-05-21 with total page 174 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book T Cell Protocols written by Kelly P. Kearse and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 357 pages. Available in PDF, EPUB and Kindle. Book excerpt: The purpose of T Cell Protocols: Development and Activation is to c- lect a series of protocols, particularly those that have been developed within the past few years, to help investigators master new techniques (or improve existing ones) for the study of T-cell Biology. Invariably, in putting together a book like this it is difficult to decide which methods to include and which to leave out. To this end methods were selected from a variety of disciplines, including cellular immunology, b- chemistry, and molecular biology, to try to provide something of interest for everyone who works on T-cell development and activation. I would like to mention that my primary reason for agreeing to put this book together is that, when I was a graduate student, I purchased a copy of Selected Methods in Cellular Immunology by Mishell and Shigii which proved a tremendous help in learning the basics of one-and two- dimensional gel te- niques (and other methods). The cover has long since fallen off, but it still remains one of my most valued reference books for the laboratory. It is my hope that T Cell Protocols: Development and Activation will prove similarly useful to current and future scientists wishing to learn new methods for expl- ing the development and activation of T cells.
Download or read book Microfluidic Single cell Technologies for Assaying Lymphocyte Interactions written by Burak Dura and published by . This book was released on 2015 with total page 144 pages. Available in PDF, EPUB and Kindle. Book excerpt: Immune cells do not live in isolation but interact to coordinate their many actions. One of the chief routes they foster communication is through direct physical interactions that enables them to read and interpret signals mediated at membrane interfaces. Despite the critical importance of these direct interactions in determining crucial developmental and functional immunological responses, their dynamic nature together with vast heterogeneity and polyfunctionality of individual immune cells have presented technical challenges for their systematic investigation. In particular, only limited tools are available that can exert control over the individual cells and their microenvironments to be able to precisely define interactions and deeply profile their outcomes at the individual cell level to resolve emerging immune responses within each single-cell. To fill this critical void, this thesis presents the development and implementation of novel microfluidic technologies for single-cell analysis of direct cell-cell interactions in immunology. By combining carefully designed weir-based hydrodynamic traps with a multistep cell loading procedure, the microfluidic devices capture and controllably pair hundreds of cells in parallel. This approach provides requisite control over interactions with one-to-one interacting partners, well-defined and synchronous initiation of interactions, and enduring contacts. It also provides full control over the soluble microenvironment by solution exchange without losing cell registration. Accordingly, these features enable monitoring and assaying lymphocyte interactions longitudinally from the beginning with multiparametric single-cell measurements. These capabilities in turn allow probing into complete immune cell activation window from the very onset for direct correlation analyses within hundreds of individual cells in a single experiment. We apply these new 'microfluidic cell pairing' technologies to quantitative investigation of lymphocyte interactions to elucidate lymphocyte activation dynamics and their relation to diverse functional behaviors at the single-cell level. These studies help resolve qualitatively and quantitatively distinct calcium signaling patterns in single CD8 T cells based on varying T cell receptor affinities which correlate with differential cytokine output. Similar studies with natural killer (NK) cells identify a previously unreported inverse correlation between the strength of early calcium signaling and cytokine production, and further indicate a calcium-dependent mechanism for selective regulation of cytotoxicity and cytokine production in NK cells. Collectively, these findings provide essential insight into the regulation and evolution of immune responses within individual immune cells, and establish the potential of these new microfluidic technologies to address important questions on many aspects of cell-cell interactions across biology in general and in immunology in particular.
Download or read book Microfluidic Devices for Studying Early Response of Cytokine Signaling written by Linlin Ye (Ph. D.) and published by . This book was released on 2008 with total page 125 pages. Available in PDF, EPUB and Kindle. Book excerpt: (Cont.) In this study, a new microscope stage holder was designed and machined, and an auto cell counting algorithm was developed for the single-cell analysis. This single-cell method provided data on cell-to-cell variations and showed that the average cell signaling profiles were consistent with those by population-based analysis. The integration of the single-cell imaging and microfluidic method enabled measurements shows promise as a technique for exploring the cell signaling with single-cell resolutions.
Download or read book Diagnostic Devices with Microfluidics written by Francesco Piraino and published by CRC Press. This book was released on 2017-06-26 with total page 281 pages. Available in PDF, EPUB and Kindle. Book excerpt: Focuses specifically on diagnostic applications. Explores the commercial aspects of developing microfluidic diagnostic device. Highlights the growing field and presents a selection of important topics making it an excellent introductory reading for graduate students in bioengineering and related disciplines. Teaches the reader how to fabricate, apply, and market microfludic diagnostic chips for lab and at home use. Discusses patient-focused development of diagnostics devices.
Download or read book Development of a Novel Microfluidic Platform to Study T Cell Signaling written by Shannon L. Faley and published by . This book was released on 2007 with total page 109 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Microfluidic Modules for Enabling Point of Care Biopsy based Cancer Diagnostics written by Debkishore Mitra and published by . This book was released on 2013 with total page 144 pages. Available in PDF, EPUB and Kindle. Book excerpt: The analyses of patient tumor biopsy samples and biopsy-based diagnostics have emerged as important tools for cancer diagnostics. However, the techniques employed currently are restricted to centralized laboratories as they are time-consuming, manual labor intensive and vary considerably in their effectiveness amongst institutions and countries. Point of Care testing (POCT) for cancer with the capacity for multiplexed detection of numerous biomarkers in biopsy samples in a rapid, precise and portable manner is emerging as an area with enormous potential to disseminate universal diagnostics to cancer patients. Additionally, POCT can be used as a screening tool, to discern malignant from benign tumors at the physician's office, and lead to reduction in the need for expensive and time-consuming laboratory tests, hence minimizing the cost and anxiety, for patients with benign tumors. A POCT platform for multiple biomarker analysis can not only improve the operational characteristics of assays but can also help ascertain drug efficacy, ushering in personalized medicine for the patients. The reduced volumes and diffusion distances, which enable multiplexed, portable and quick assays, in microfluidic devices makes such devices a promising platform to realize POCT systems. But current microfluidic devices for cancer diagnostics suffer from the lack of a generalized on-chip sample preparation module and a simplified fluid actuation technique. The overall goal of the reported dissertation research is to develop microfluidic modules that will enable the development of integrated microfluidic diagnostic platforms for the multiplexed detection of cancer biomarkers in tumor biopsy samples. The main focus of the thesis is on the development of novel microfluidic sample preparation modules. The purpose of the sample preparation component is to pre-concentrate cancerous cells, remove background proteins in the sample and to subsequently lyse the cells to release the proteins of interest. The pre-concentration of the adherent cells, including the cancerous cells, in the sample is reported by trapping them using a novel hydrodynamic cell trap. The sample washing methods, to remove extracellular proteins that could interfere with downstream assays, is also optimized. An electrochemical lysis technique is then integrated to the cell pre-concentration module, to effectively lyse the cells without having to add external reagents. Microfluidic modules for the separation of bacterial and mammalian cells from mixed samples are also reported. The immortalized cancer cell lines used in this research include the human breast cancer cell lines BT-474, known to over express the Her-2 protein, and T47D along with cervical cancer cell line HeLa. The development of a novel fluid actuation technique, termed Proximal Degas-driven Flow (PDF), is also reported in this thesis. PDF takes advantage of the high porosity and air solubility of PDMS to reduce the pressure inside the fluidic channels leading to fluid flow in the channel. This actuation technique enables bubble-free fluid flow, can be used to fill up dead-end chambers in contrast to traditional pressure (positive or negative) driven flows and does not require the priming of the channels. Unlike degas-driven flow, PDF alleviates the need for pre-degassed and sealed devices, enabling consistent and longer-lasting fluid flow. This portable technique also requires very simple and cheap hardware like a vacuum bulb or membrane pump (thumb pump). In conclusion, several microfluidic modules to enable Point of Care biopsy-based cancer diagnostics are introduced. The research presented in this dissertation is an effort to transform point-of-care cancer testing and provide universal diagnostics and personalized medicine to cancer patients.
Download or read book Microfluidic Organ on a Chip Revolutionary Platforms for Disease Comprehension and Treatment written by Shi-Cong Tao and published by Frontiers Media SA. This book was released on 2022-01-11 with total page 176 pages. Available in PDF, EPUB and Kindle. Book excerpt: Existing culture systems have a limited ability to reproduce the complicated and dynamic microenvironment of a functioning organ. To solve the issues of conventional culture techniques, multidisciplinary researchers, involving medical doctors, stem cell and developmental biology experts, engineers and physical scientists, have emerged to innovate methods and devices. A microfluidic organ-on-a-chip (μOOC) is a cell culture device, based on microfluidic technology, which contains continuously perfused chambers with cells to simulate organ-level physiology/pathology. The μOOC is not to build a whole living organ, but rather to synthesize minimal functional units that recapitulate organ-/tissue-level functions. The μOOC can be applied to study not only the convention stimulation on cells by molecular/drugs, but also physical forces (fluid shear stress, cyclic strain and mechanical compression), organ-specific cell-cell intercommunication, and organ-organ coupling responses. There is an emerging need for innovative approaches for the production, control, analysis, and utilization of the μOOC, and even the multiple interconnected μOOC (Human-on-a-Chip). Although the μOOC has attracted much attention and is continuous being studied, there are still many difficult problems to be solved. Some of the most mentioned challenges include microenvironmental (biochemical, biophysical, biomechanical, nutrient, etc.) control, modeling tissue–tissue and multiorgan interactions, and reducing variability (automated control, high-throughput manipulation/analysis, integration of biosensing and etc.).
Download or read book Microfluidics and Biosensors in Cancer Research written by David Caballero and published by Springer Nature. This book was released on 2022-06-27 with total page 599 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book offers a comprehensive overview of the development and application of microfluidics and biosensors in cancer research, in particular, their applications in cancer modeling and theranostics. Over the last decades, considerable effort has been made to develop new technologies to improve the diagnosis and treatment of cancer. Microfluidics has proven to be a powerful tool for manipulating biological fluids with high precision and efficiency and has already been adopted by the pharmaceutical and biotechnology industries. With recent technological advances, particularly biosensors, microfluidic devices have increased their usefulness and importance in oncology and cancer research. The aim of this book is to bring together in a single volume all the knowledge and expertise required for the development and application of microfluidic systems and biosensors in cancer modeling and theranostics. It begins with a detailed introduction to the fundamental aspects of tumor biology, cancer biomarkers, biosensors and microfluidics. With this knowledge in mind, the following sections highlight important advances in developing and applying biosensors and microfluidic devices in cancer research at universities and in the industry. Strategies for identifying and evaluating potent disease biomarkers and developing biosensors and microfluidic devices for their detection are discussed in detail. Finally, the transfer of these technologies into the clinical environment for the diagnosis and treatment of cancer patients will be highlighted. By combining the recent advances made in the development and application of microfluidics and biosensors in cancer research in academia and clinics, this book will be useful literature for readers from a variety of backgrounds. It offers new visions of how this technology can influence daily life in hospitals and companies, improving research methodologies and the prognosis of cancer patients.
Download or read book The Initial Events of T cell Activation in Realistic Model Systems written by Markus Koerbel and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Spatiotemporal Receptor Dynamics During Early T Cell Signaling written by Nicole Cheung Fay and published by . This book was released on 2014 with total page 65 pages. Available in PDF, EPUB and Kindle. Book excerpt: A given T cell receptor (TCR) can robustly discern a pathogen-derived agonist peptide amidst a myriad of background peptides, all bound to major histocompatibility complexes (MHC). This remarkable degree of discrimination is the culmination of physical operations happening at the membrane-membrane junction between a T cell and an antigen-presenting cell. In the research described in this dissertation, I applied optical methods to hybrid interfaces between a live primary T cell and a supported lipid bilayer mimicking an antigen-presenting cell (APC) surface. In this manner, I revealed a number of novel mechanisms by which ligand-receptor dynamics dictate T cell signaling output. First, a two-parameter titration of two ligands - agonist peptide-MHC and the costimulatory surface molecule CD80/B7-1 - revealed that the density of CD80 influences the TCR activation threshold. Additionally, co-presentation led to an interdependent trafficking scheme of these surface molecules that may serve to boost the effectiveness of CD80 costimulation at low agonist peptide-MHC densities and reduce spurious activation under other conditions. Second, it was possible to resolve TCR microclusters by size using a nanoparticle array embedded in the ligand-presenting bilayer. This innovative form of size-based membrane-receptor chromatography in live cells revealed that the maximal size of the TCR microclusters was regulated by engagement with MHC molecules occupied by our model agonist peptide (moth cytochrome c). T cell antigen recognition and subsequent activation was found to be unaffected by the percolation of actively signaling TCR microclusters through this nanoparticle array. Third, myosin activity was responsible for the rapid centripetal burst of TCR microclusters in the initial 60 seconds after antigen exposure. Importantly, inhibition of myosin-induced forces abolished T cell activation, a process potentially mediated by the force-tension sensor CasL. In summary, T cell response potency results from spatiotemporal coordination of a massive interconnected signaling network undergoing continuous feedback with ligand-receptor binding events.
Download or read book Real time Cell Secretion Analysis Inside Microfluidic Devices written by Kyungjin Son and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Cells secrete various biomolecules, including cytokines or growth factors and enzymes, to mediate cellular behavior and cell-cell signaling in the immune system, tumor microenvironment, or stem cell niche. It is essential to analyze cell-secreted products to understand how cells response to external stimuli, which may provide us valuable information and knowledge related to basic cell biology, disease diagnosis and therapeutics. This dissertation describes the development of microfluidic-based cell culture microsystems with integrated biosensors for monitoring of cellular secretory activities in real-time, which aims at better understanding of cancer cellular signaling to mediate the extracellular matrix in cancer cell invasion and metastasis, cellular inflammatory response upon mitogenic stimulation, and autocrine signaling via multiple growth factor release. Various approaches, such as utilization of 3D hydrogel matrix for incorporating sensing elements or application of antibody-conjugated microbeads, were used to enhance the sensitivity of microsystems, which enables to detect low abundant cell-secreted molecules not only from a small group of cells but also from individual cells. Single cell studies revealed the presence of heterogeneous secretion levels of protease, cytokines and exosomes from individual cells, which may not be accessible using population-averaged assays. With a photo-initiated selective cell retrieval method, further in-depth molecular biology analyses are also achievable. Consequently, simultaneous detection of growth factors released from primary hepatocytes was demonstrated using hydrogel-incorporating microsystems for maintaining cellular phenotypes and functions similar to in vivo.
