........ published in NEWSLETTER # 49
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........ published in NEWSLETTER # 49
FLOW CYTOMETRY
by Dr. A. Jacquemin_Sablon, CNRS, Villejuif (France) and Dr. H.A. Crissmann, Los Alamos National Laboratory, Los Alamos/NM (U.S.A.)
This volume (NATO ASI SERIES H67) contains the results of the NATO ASI on `Flow Cytometry', that was designed to address three major areas of interest in cell and molecular biology: (a) cell activation and biological response, (b) membrane_ligand interactions and cell identity and (c) nuclear components form and function. Data management, expert systems and cell sorting techniques that are subjects of concern to all aspects of flow cytometry, are also presented.
Cell activation is an early event in the physiological process that elicits a response of a cell to perform some specialized function. It is known that several cytolosic events are related to cell activation. The course focused on optical techniques that can be used in flow cytometry to study the cascade of processes that are involved. One example describes the use of a peptide hormone for studying the activation of smooth muscle cells. The aim is to determine whether calcium and mitochondrial responses could be observed that are consistent with the interaction between the signal and energy transduction pathways. Methods for detecting activation_induced changes in intracellular pH and membrane potential are addressed. Although the process of cell activation is still not well understood, many of the events that occur in tandem can be studied by flow cytometry and new fluorescent probes that have recently been developed for that purpose are presented.
The capability for following the migration of specific cell types through different parts of the body has recently become available by a process that involves labeling of cell membrane with fluorescent, non_toxic probes that are not easily degraded. If cells undergo division, the fluorescent label is partitioned between the two daughter cells. In this way flow cytometric analyses can be used to `track' and follow the proliferative potential and isolation of specific subpopulations of cells. Attempts to identify the hemapoeitic stem cells in bone marrow by flow cytometry are of particular interest. Some success has been achieved and stem cells, capable of reviving a functional hemapoeitic system in lethally irradiated mice have been obtained by cell sorting. Several new FCM techniques are described for obtaining viable, cell populations for functional assays.
The numerous functional activities that occur in the cell nucleus in response to intra and intercellular signals have invoked a large series of flow cytometric techniques. Changes in chromatin structure in response to cell cycle or physiological_related changes can be analyzed with metachromatic fluorochromes such as acridine orange following acid or heat denaturation of cells. Rates of DNA synthesis are also examined by analysis of BrdU_incorporation into DNA and subsequent flow cytometric analysis of cellular BrdU content using fluorescein_labeled antibodies to BrdU. Chromosome analysis and sorting of chromosomes from metaphase arrested cells
are now possible. Techniques are described for performing fluorescence i situ hybridization (FISH) on sorted chromosomes. Applications of PCR and gene mapping on sorted chromosomes are also now feasible. The advanced flow cytometry course provided the most up_to_date techniques available at present.
Reference books: A88, H58, E156, H67