Flow Cytometry With Plant Cells

Targeted at beginners as well as experienced users, this handy reference explains the benefits and uses of flow cytometery in the study of plants and their genomes. Following a brief introduction that highlights general considerations when analyzing plant cells by flow cytometric methods, the book goes on to discuss examples of application in plant genetics, genomic analysis, cell cycle analysis, marine organism analysis and breeding studies. With its list of general reading and a glossary of terms, this first reference on FCM in plants fills a real gap by providing first-hand practical hints for the growing community of plant geneticists.

This work present practical, biotechnological applications of flow cytometry techniques for the study of animal, plant and microbial cells, explaining methodologies for sample preparation, staining and analysis. It discusses cell variability in cell culture processes and shows how the quantitative analysis of heterogeneous populations aids in the biotechnological exploitation of cells.

Many studies in both plant and animal systems have shown that Matrix Attachment Regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. MARs are AT-rich sequences of DNA that bind in vitro to the proteinacous filament-like structure within the nucleus called the nuclear matrix. In our investigation of transgenic Nicotiana tabacum NT-1 cells in culture, we have observed that transgene expression is often variegated. In other words, some cells in an isogenic population do not express the transgene, and/or other cells within the same population express the transgene at varying levels. The question was raised: Do MARs increase transgene expression by altering variegation? More specifically, do MARs increase the percentage of cells expressing the transgene, increase the magnitude of expression in expressing cells, or both? In order to address these questions, it was necessary to quantitate transgene expression variegation at the resolution of individual cells. We chose to measure Green Fluorescent Protein (GFP) expression in individual tobacco NT-1 cells by flow cytometry. In order to analyze individual cells and because NT-1 cells in culture grow as filaments, it is necessary to prepare protoplasts that can pass one at a time through the flow cell of the flow cytometer. We found that current flow cytometry methods for measuring GFP expression in plants were susceptible to debris resulting from protoplast preparations. We observed that when the plasma membrane of protoplasts is breached, GFP diffuses out into the medium, and flow cytometric measurements of these non-viable protoplasts imply they do not express GFP. This debris can overestimate the proportion of non-expressing cells in the population. In order to correct this problem, we used an approach called a dye exclusion test. Because propidium iodide enters protoplasts to stain nuclei only when the plasma membrane is breached, debris that stains with this dye can be removed from.

Shows how flow cytometry is integrated into modern biotechnology. This volume deals with issues of content, sensitivity, and high throughput informatics with applications in genomics, proteomics and protein-protein interactions, drug discovery, vaccine development, plant and reproductive biology, pharmacology and toxicology, and more.

"Flow Cytometry - Recent Perspectives" is a compendium of comprehensive reviews and original scientific papers. The contents illustrate the constantly evolving application of flow cytometry to a multitude of scientific fields and technologies as well as its broad use as demonstrated by the international composition of the contributing author group. The book focuses on the utilization of the technology in basic sciences and covers such diverse areas as marine and plant biology, microbiology, immunology, and biotechnology. It is hoped that it will give novices a valuable introduction to the field, but will also provide experienced flow cytometrists with novel insights and a better understanding of the subject.

The aim of this volume is to merge classical concepts of plant cell biology with the recent findings of molecular studies and real-world applications in a form attractive not only to specialists in the realm of fundamental research, but also to breeders and plant producers. Four sections deal with the control of development, the control of stress tolerance, the control of metabolic activity, and novel additions to the toolbox of modern plant cell biology in an exemplary and comprehensive manner and are targeted at a broad professional community. It serves as a clear example that a sustainable solution to the problems of food security must be firmly rooted in modern, continuously self re-evaluating cell-biological research. No green biotech without green cell biology. As advances in modern medicine is based on extensive knowledge of animal molecular cell biology, we need to understand the hidden laws of plant cells in order to handle crops, vegetables and forest trees. We need to exploit, not only empirically, their astounding developmental, physiological and metabolic plasticity, which allows plants to cope with environmental challenges and to restore flexible, but robust self-organisation.

The current technology and its applications in flow cytometry are presented in this comprehensive reference work. Described in explicit detail are the instrumentation and its components, and applications of the technology in cell biology, immunology, pharmacology, genetics, hematology and clinical medicine. Methods for data analysis, including both hardware and software, and explicit experimental techniques for making specific measurements are presented. Material is divided by topic into two volumes: Volume I covers instrumentation, genetics, and cell structure; Volume II contains material on cell function studies by flow cytometry. This reference is essential for both the novice and the experienced investigator using flow cytometry in research, and for students of cell biology, biomedical engineering, and medical technology.

From the Reviews of the First Edition: "This is a good reference manual for multi-user facility faced with a wide variety of biological applications." - CYTOMETRY "Flow Cytometry includes an impressive array of methods applicable to chromosome analysis, plant biology, marine biology, fluorescence, insitu hybridization, and others. It succeeds in providing the reader with good insight into the power of the technology throughout biology." - KENNETH A. AULT, MAINE CYTOMETRY RESEARCH INSTITUTE, MAINE MEDICAL CENTER, IN CANCER CELLS Flow Cytometry, Second Edition provides a complete and comprehensive two-volume laboratory guide and reference for the use of the most current methods in flow cytometry sample preparation and analysis. These essential techniques are described in a step-by-step format, supplemented by explanatory sections and trouble-shooting tips. The methods are accessible to all researchers and students in biomedical science and biology who use flow Cytometry to separate and analyze cells. * * Comprehensive methodological coverage in unique style * In depth treatment of procedures * Description of each procedure's: * Theoretical foundations * Critical aspects * Possible pitfalls * Written by authors with extensive experience who: * Developed or modified the technique * Describe their experience with different instruments and applications to different cell systems * Are the Who's Who in Flow Cytometry * 10 methods cover assessment of apoptosis and other modes of cell death * Practical, handbook-style presentation works in lab or classroom * Printed on acid-free paper * Color plates